The endosperm is not a uniform tissue. The cellularized endosperm contains several cell types based on expression patterns and cytological differences. These structures are arranged along the axis running from the anterior pole where the pollen tube delivers the sperm cells (the micropyle) to the posterior pole where maternal nutrients transit (the chalaza). Other cell types are arranged according to radial symmetry. These two axes of patterning are conserved amongst flowering plants.
1. Polar organization
An antero-posterior axis pre-exists in the embryo sac and it is not known if this axis influences the corresponding MC axis in the endosperm. The micropyle is the site of entry of the pollen tube in the ovule. The chalazal pole is opposite the micropyle and represents the area where the maternal nutrients are deposited in the endosperm. In many species, including A. thaliana and maize (Zea mays), this axis is curved, such that the micropyle and the chalaza become positioned side by side at the end of ovule development. During the syncytial stage, the influence of the MC axis is observed in the orientation of syncytial divisions, the mitotic domains and the migration of NCDs (Fig. 2). The first two divisions take place along the MC axis of the endosperm. The third division is perpendicular to the MC axis. Subsequently, the mitotic domains are established first in the chalazal endosperm (CZE), with endoreduplication replacing nuclei division. Later nuclei division in the micropylar endosperm (MCE) no longer takes place in synchrony with divisions in the peripheral endosperm (PEN) (Fig. 2). This general organization is also marked by a distinct cytological organization of the cytoskeleton in the three domains (Brown et al., 1999; Nguyen et al., 2002). The CZE, initially defined by two to four large nuclei in a pool of cytoplasm forming the cyst, is later marked by a posterior-directed migration of NCDs (Guitton et al., 2004). After developmental stage VI, each syncytial division is followed by gradual migration of NCDs from the posterior part of the peripheral endosperm toward the cyst. Theses NCDs fuse to generate multinucleate nodules, which appear as large NCDs (Fig. 2). Over time, individual NCDs and nodules migrate and fuse with the chalazal cyst. As cellularization takes place, all remaining NCDs fuse into the cyst, which gradually recedes, while the entire peripheral endosperm becomes cellular.
The identity of the three domains along the MC axis is established by specific expression of genes and other markers. In A. thaliana, the micropylar endosperm, which occupies a domain called the embryo-surrounding region (ESR), is marked by the expression of GFP in the enhancer trap line N9185 (Ingouff et al., 2005b), the sucrose transporter AtSUC5 (Baud et al., 2005), the subtilisin-like serin protease ABNORMAL LEAF SHAPE 1 (Tanaka et al., 2001), the Basic helix loop helix factor ZHOUPI (Yang et al., 2008), the transcription factor MINISEED3 (Wang et al., 2010a) and several other genes that remain to be catalogued from an extensive transcriptome study of dissected seed components (Le et al., 2010). In cereals, several other genes have been characterized by their localized expression at the embryo-surrounding pole (Cossegal et al., 2007) and at the posterior (basal) pole (Royo et al., 2007). The MC axis is thus potentially conserved but homologous functions in the various domains between cereals and A. thaliana remain to be identified. The three domains of the A. thaliana endosperm have been dissected and the transcriptome analyzed at different developmental stages (Le et al., 2010).
The definition of MC polarity is compromised in mutants for the polycomb group (PRC2) pathway FIS, including MEDEA (MEA), FERTILIZATION INDEPENDENT SEED 2 (FIS2), FERTILIZATION INDEPENDENT ENDOSPERM (FIE), MULTICOPY SUPPRESSOR OF IRA 1 (MSI1) and DEMETER (Grossniklaus et al., 1998; Luo et al., 1999, 2000; Choi et al., 2002; Guitton & Berger, 2005). The expression of MEA and FIS2 is limited to the endosperm, providing a specific tissue identity for the FIS polycomb group complex (Luo et al., 2000; Wang et al., 2006), while FIE and MSI1 are expressed in other tissues (Yadegari et al., 2000; Hennig et al., 2003) and participate in other PRC2 protein complexes (Chanvivattana et al., 2004; Katz et al., 2004).
Mutations in FIS genes disrupt mitotic domain organization (Ingouff et al., 2005b). The FIS complex also prevents the down-regulation of AGL62 expression (Kang et al., 2008), presumably leading to the absence of cellularization in fis mutant endosperm (Chaudhury et al., 1997; Kiyosue et al., 1999; Sorensen et al., 2002). FIS genes presumably control the transition between developmental phases during endosperm development and fis loss of function prevents exit from the juvenile stage before the definition of mitotic domains. This hypothesis has been supported by genome-wide analyses (Weinhofer et al., 2010). Using tagged fluorescent endosperm nuclei it was possible to isolate a fraction enriched in endosperm chromatin and to study the genome-wide profile of H3K27me3. The repressive marks H3K27me3 were present over a large set of genes that are not expressed during early endosperm development, suggesting that the FIS PcG prevents the acquisition of somatic traits in the endosperm and thereby preserves the endosperm identity. A group of genes specifically expressed during early wild-type endosperm development became repressed around the heart stage by the FIS complex (Weinhofer et al., 2010), supporting the idea that the FIS PcG complex is required for the repression of a defined set of genes around endosperm cellularization (Ingouff et al., 2005b; Berger et al., 2006). Beside this global molecular control by the FIS complex, the molecular and cellular mechanisms responsible for the definition of the mitotic domains in the syncytial endosperm are not understood.
2. Radial organization
After cellularization, the outer endosperm cell layer undergoes a specific series of events leading to its differentiation into a specialized structure, the aleurone layer (Becraft & Yi, 2011). In cereals, the aleurone layer shows specific storage of phytic acids that chelate mineral ions such as magnesium and phosphate. Aleurone cells are involved in cereal grain maturation (Vicente-Carbajosa & Carbonero, 2005). During germination, enzymes produced by aleurone cells are important for remobilization of the reserves of the starchy endosperm. This differentiation has been documented in detail in cereals (Becraft, 2007). After cellularization, repetitive periclinal divisions form cell files, and the outermost of these cells undergoes a series of anticlinal divisions leading to the aleurone layer of cuboidal cells. Defective endosperm kernel 1 (Dek1), encoding a cysteine proteinase (Lid et al., 2002), and Crinkly 4 (Cr4) (Becraft et al., 1996; Becraft & Asuncion-Crabb, 2000), encoding a leucine-rich repeat kinase, are important for aleurone differentiation in maize. The function of these genes is controlled or regulated by Supernumerary aleurone layer 1 (Sal1), which may be involved in receptor recycling (Shen et al., 2003). The signaling from Dek1 is mediated by the locus defining the negative regulator Thick Aleurone 1 of unknown identity (Yi et al., 2011). Phylogenetic and functional analyses of the four genes encoding serine/threonine receptor kinases in rice (Oryza sativa) and A. thaliana identified an ortholog in rice but not in A. thaliana, suggesting that this family acquired different functions in endosperm development in dicots and monocots (Cao et al., 2005). The Dek1 homolog in A. thaliana may have a general role in cell differentiation and the disorganized outer endosperm cell layer in dek1/dek1 seeds does not necessary reflect a specific function in aleurone differentiation (Lid et al., 2005). In conclusion, it remains undetermined whether the aleurone layer in the cereal endosperm is equivalent to the outer endosperm layer in Arabidopsis.
In cereals, cells of the future aleurone layer that are adjacent to the site of delivery of maternal nutrients through the nucellus differentiate as transfer cells (TCs). TCs are very elongated, develop cell wall in-growths and express sugar and amino acid transporters (reviewed in Royo et al., 2007). TCs are conspicuous in maize, less numerous in wheat (Triticum aestivum) and barley (Hordeum vulgare) and hardly distinguishable in rice. These cells are responsible for the intake of maternally provided nutrients. TCs also express cysteine-rich polypeptides involved in defense (Gomez et al., 2009). Maize TCs express MYB-related protein 1 (ZmMPR-1), which activates expression of several genes (Gomez et al., 2002). This transcription factor is sufficient to cause TC differentiation from other domains of the aleurone, suggesting that TC differentiation results from a sequential series of activations that take place locally and redirect aleurone differentiation (Gomez et al., 2009). The signals that cause this local activation of ZmMPR-1 expression are unknown but they could involve sensing sugar or other products delivered from the phloem.