**2. Synthesis of storage reserves during seed filling**

The main carbon source for biosynthesis and nutrient accumulation in seeds is sucrose, which is produced by the products of photosynthesis in plants. Sucrose is transported from the vegetative parts where photosynthesis occurs to the developing seeds. In cereals and legumes, nitrogen is transported to seeds mainly in the form of asparagine and glutamine; in some species, alanine may also serve as a nitrogen source [1]. Cerelas obtain nitrogen from organic or chemical fertilizers. Ureides, allantoin, and allantoic acids are the major forms of organic nitrogen transport in legumes [2, 3], and about 10–15% of organic nitrogen is transported as ureides in soybean and cowpea. Nitrogen-fixing symbiotic *Rhizobium* in nodules produce ammonium used for purine and uric acid synthesis, and uric acid is transported to neighboring uninfected cells to synthesize allantoin in peroxisomes [4].

During seed filling, carbohydrates are constantly transported from vegetative parts, so the conversion and accumulation of photosynthetic products varies greatly among plant species. In wheat and barley, the net photosynthetic activity in the flag leaf and spike is quite high, while the sugar produced in the leaves below the tassel makes a lower photosynthetic contribution to the grain in maize. However, sugars produced in the leaves enveloping the cob are efficiently transported to the developing seeds. In legumes, sucrose is deposited in the form of starch in the leaves and pods, and nitrogen is stored in the leaves and remobilized to the developing seeds.

Vascular tissues transport nutrients and water and terminate in the placentochalazal region and seed coat of monocotyledons and dicotyledons, respectively. Nutrients are transported and released into the apoplast and taken up by the developing endosperm and embryo during seed filling. Vascular tissues across the developing grain facilitate the transport of nutrients to the endosperm in winter cereals. Specialized transfer cells facilitate the transport of nutrients from the pedicel to the endosperm in warm-season cereals. In legumes, reserves are accumulated in the cotyledons and nutrients are transported via vascular tissue to the funiculus, from where they enter the apoplast space and are then redistributed in the developing seed.

Seeds must have long-term energy stores in the form of starch, lipids, or hemicellulose to ensure successful germination and seedling development. These carbon sources are stored in the cotyledons or endosperms of most plant species. The conversion of assimilated carbon, usually in the form of sucrose, into various storage compounds in different tissues is regulated by complex interactions of gene expression and metabolic activity during seed development [5–7]. Starch is present in most

#### *Seed Filling DOI: http://dx.doi.org/10.5772/intechopen.106843*

plant tissues as a carbon storage compound and can account for up to 70% of the dry weight of seeds in many cereal grains [8].

The structure and composition of starch, which is inert and insoluble in water, makes it an ideal storage material that allows large amounts of sugars to be stored in cells without negatively affecting the dissolution potential in seeds. Starch accumulation begins in endosperm cells shortly after fertilization and rapid cell division [9, 10]. The number of endosperm cells can be used as an indicator of yield. The cell division phase is completed within 2–6 days after pollination (DAP), but cell volume continues to increase until maturity (~35–40 DAP) [11]. Accumulation of storage reserves usually occurs between 10 and 35 DAP [1].

#### **2.1 Starch synthesis**

There are at least four groups of enzymes involved in starch synthesis in plants. They are ADP-glucose pyrophosphorylases (AGPase), starch synthases (SS), starch branching enzymes (SBE) and starch debranching enzymes (DBE) (**Figure 1**). Plants usually have several isozymes of each group, 14 forms of these enzymes (2 AGPase, 5 SS, 3 SBE ve 4 DBE) are involved in starch synthesis and 13 of them show varying degrees of homology in all plans [13]. Sucrose is used as a substrate for starch formation to produce straight-chain amylose and branched amylopectin in seeds (**Figure 1A**). In cell cytosols, sucrose is converted to fructose (Fru) and uridine diphospho glucose (UDPG1c) by sucrose-UDP glucosyltransferase.

Fructose is phosphorylated by hexose phosphate isomerase to Glc-6-P and to Fru-6-P, which is converted to G1c-1-P by phosphoglucomutase. UDPG1c is also converted to Glc-1-P by UDPG1c pyrophosphorylase (UGPase). The first step of starch synthesis is the formation of ADP-glucose by AGPase [14]. The reaction catalyzed by AGPase is the first stable step in the biosynthesis of both temporarily stored starch in chloroplasts and chromoplasts and starch stored in amyloplasts. This enzyme is located in the plastids of photosynthetic tissue and has different forms in seeds, therefore its cellular location may vary in different plants. While most of the AGPase is found in the plastids of potato and pea, it is mainly located in the cytoplasm in maize, barley, and rice [15]. The enzyme carries out the following reaction: Plastid alkaline conversion of inorganic pyrophosphate (PPi) to inorganic phosphate (Pi) maintains the balance in favor of ADP-glucose synthesis through the action of inorganic pyrophosphatase [16], which can be transported through the plastid envelope [17]. The conversion of ADP-glucose occurs in the amyloplasts of storage cells of dicotyledons (**Figure 1**), while it occurs in the cytosols and plastids of endosperm cells of cereals (**Figure 1**). APGase is a heterotetrameric protein consisting of two large (APG-L) and two small (APG-S) subunits encoded by two different genes [18]. Plastidial AGPase is found in all starch-synthesizing tissues, but there are at least two different AGPases, corresponding to plastidial and cytosolic isoforms of AGPase present in the developing endosperm of maize [19], barley [20], rice [21], and wheat [22]. Starch synthesis by cytosolic AGPase depends on PPi-consuming reactions catalyzed by fructose-6-phosphate, 1-phosphotransferase, and UDP-glucose pyrophosphorylase for starch biosynthesis [23, 24]. The cytosolic AGPase isoform is responsible for 65–95% of the total AGPase activity in the developing cereal endosperms. Consequently, most starch biosynthesis occurs through the import of ADP-glucose in exchange for ADP, which is a byproduct of starch synthase in plastids [25, 26]. Starch biosynthesis in non-graminaceans depends on plastidial AGPase and the import of ATP and hexose phosphates from the cytosol (**Figure 1**). The forms and activity of APGases can vary in different parts of the

#### **Figure 1.**

*Biosynthesis of starch in monocotyledons (A) and dicotyledons (B). (A) Monocotyledons have the cytosolic form of AGPase. ADP-glucose is taken up from the cytosol via the ADP-glucose/ADP transporter pathway (Bt1). (B) Hexose-phosphates and ATP are transferred from the cytosol into the plastid via the Glc 6-P/Pi antiporter (1) and the ATP/ADP transporter (2), which are located in the plastid inner envelope membrane. Cytosolic and plastidial isoforms of phosphoglucomutase (PGM) convert Glc 1-P and Glc 6-P into each other. Pi produces the pyrophosphate produced by AGPase. ADP is produced as a byproduct of starch synthase activity (SS). The starch synthases use ADP-glucose to form amylopectin with starch branching and debranching enzymes [12].*

same plant because there are several genes encoding large and small subunits of APGases [27–29]. The APG-L subunits have specific expression patterns in different tissues, such as leaves, roots and endosperm of cereals [28, 30–32], or their expression levels are regulated under specific conditions, such as sugar content in potato [33, 34].

Starch synthases catalyze ADP-glucose to glucans and these are eventually used to synthesize water-insoluble amylose and amylopectin. Granule-bound starch synthases (GBSSI and GBSSII) produce and extend the amylose chain [35, 36]. Plants carrying a mutant form of GBSS corresponding to the waxy gene in cereals, do not produce amylose. They may also be involved in the elongation of glucans in various plants such as rice [37]. Another group of starch synthase enzymes (SSI-SSIV) is encoded by several genes and plays different roles in the formation of amylopectin in different tissues and developmental stages [38]. SSI produces short glucan (Glc) chains (<10). The first synthesized amylopectin is water-soluble and is elongated by SSII and SSIII to form water-insoluble amylopectin. Besides starch, there are other types of carbohydrates in the seeds of cereals
