**2. Overview of the starch biosynthesis and degradation in plants**

Polyglucans are the most important and widespread carbohydrate storage compounds found in nature, with glycogen and starch being the most abundant forms. Both polysaccharides are comprised of glucose chains linked by an α-(1,4) bond, and branched at α-(1,6). Glycogen is a homogeneous water-soluble polymer with relatively uniformly distributed branches [22] and is found in organisms such as archaea, bacteria and certain eukaryotes. Starch is made up of amylose (a largely unbranched, minor component) and amylopectin (an asymmetrically branched major component) and is present in the cytoplasm of *Rhodophyceae* (red algae) and *Glaucophyta* [23], but is confined to the plastid stroma (chloroplasts in green tissues and amyloplasts in reserve organs) in green algae and higher plants. In fact, starch synthesis is restricted to the *Archaeplastida*, whose origins are thought to be via a single endosymbiotic event involving ancestors of cyanobacteria and a heterotrophic host [24], rendering the organelle known as the plastid, which is capable of oxygenic photosynthesis. Recent phylogenetic studies indicate that the plastidial starch pathway is complex, and made up of genes with both cyanobacterial and eukaryotic origins [25,26], and is in sharp contrast to the lower-complexity pathway of cytosolic starch synthesis found in the *Rhodophyceae* and *Glaucophyta* [27]. Phylogenetic analysis of the enzymes of the starch biosynthetic pathway strongly suggests that the pathway was originally cytosolic (in the common ancestor of the *Archaeplastida*), and then re-directed to plastids via three discrete steps, leaving some enzymes involved in the metabolism of maltooligosaccharides (MOS) and amylopectin degradation in the cytoplasm. The three evolutionary steps involved are: (1) plastidial synthesis of unbranched MOS; (2) glycogen synthesis (including priming steps and branching activities); and (3) plastidial starch synthesis, resulting in the eventual loss of cytosolic starch synthesis. Interestingly, the relocation of the starch synthesis pathway to plastids coincides with the evolution of lightharvesting complexes [26,28].

494 The Complex World of Polysaccharides

yields up to 40% [9,14].

qualities is a challenge [5].

soybean planted was transgenic [18].

different plant species using different strategies.

been shown that the later approach leads to formation of very thermally stable RS3, and to

Finally, but not least, is the role of investigation and development conducted by researchers from universities and industry. The incorporation of progress in science and the use of currently existing technology contributes to the production of healthy foods, and in this context, designing plants with biology tools to improve their current molecular nutritional

The first use of transgenesis in plants in the 1980s brought the arrival of a powerful tool for the study of metabolic regulation and crop improvement. Of particular interest from a health and commercial viewpoint was the potential for increasing yield making alteration of carbon partitioning between sucrose, starch and amino acids [17]. Since that time, plant biotechnology and its commercialization are in exponential phase. Already In 1998, more than 28 million hectares of transgenic crop plants were grown worldwide. Of these 28 million hectares, the largest area was in the USA (22 million hectares) followed by Canada (1.8 million hectares), Argentina (1.8 million hectares) and China (estimated at 1.1 million hectares). It was also estimated that in the US 40% of the cotton, 24% of corn and 40% of

Given the large amount of information available from molecular biology studies and from genomic programs about the starch biosynthetic genes from crop plants, it is now relatively simple to identify the changes at the DNA level to generate desired starch phenotypes [19- 21]. Transgenic approaches to altering the composition of crop plants involve two general approaches: overexpression of an endogenous or foreign gene in the target tissue, and use of RNAi technology to specifically suppress the activity of a specific plant gene [10]. We propose in this chapter to give an overview of starch synthesis to review the potential target technologies and to summerize the successful work done by numerous research groups in

**2. Overview of the starch biosynthesis and degradation in plants** 

Polyglucans are the most important and widespread carbohydrate storage compounds found in nature, with glycogen and starch being the most abundant forms. Both polysaccharides are comprised of glucose chains linked by an α-(1,4) bond, and branched at α-(1,6). Glycogen is a homogeneous water-soluble polymer with relatively uniformly distributed branches [22] and is found in organisms such as archaea, bacteria and certain eukaryotes. Starch is made up of amylose (a largely unbranched, minor component) and amylopectin (an asymmetrically branched major component) and is present in the cytoplasm of *Rhodophyceae* (red algae) and *Glaucophyta* [23], but is confined to the plastid stroma (chloroplasts in green tissues and amyloplasts in reserve organs) in green algae and higher plants. In fact, starch synthesis is restricted to the *Archaeplastida*, whose origins are thought to be via a single endosymbiotic event involving ancestors of cyanobacteria and a heterotrophic host [24], rendering the organelle known as the plastid, which is capable of oxygenic photosynthesis. Recent phylogenetic studies indicate that the plastidial starch There are four biochemical steps in each tissue that are required for the synthesis of starch, substrate activation, chain elongation, chain branching, and chain debranching [10] and it involves at least three enzymes such as ADP-glucose pyrophosphorylase (ADPGlc PPase, EC 2.7.7.27), starch synthase (SS, EC 2.4.1.21), and branching enzyme (BE, EC 2.4.1.18) [29,30] (Figure 1).

The first step of the starch biosynthesic pathway is the synthesis of the activated monomer ADPglucose (ADPGlc) from glucose-1-phosphate and ATP, synthesized by ADPGlc PPase. This reaction is the key step for the control of carbon flux through the starch biosynthetic pathway [29,30].

The second step of the starch biosynthesis pathway is the reaction catalyzed by starch synthase, in which the glucosyl moiety of ADPGlc is transferred to the non-reducing end of a pre-existing α-1,4 glucan polymer [10]. To date, five SS isoforms have been described based on sequence similarities: granule-bound SS (GBSS), involved mainly in amylose synthesis and the soluble isoforms: SSI (involved in the synthesis of small chains of amylopectin), SSII and SSIII (with a major role in amylopectin synthesis) and SSIV (recently found to be involved in the control of starch granule number and starch granule initiation) [31-34].

To produce an efficient clustering of the branch points and the formation of crystalline lamella, several debranching enzymes (DBE) are required [35,36]. In addition, the degradation of the crystalline granules depends on a recently discovered group of enzymes – the glucan, water dikinases (GWDs) – which phosphorylate crystalline sections of the granules. Such phosphorylation is catalyzed by two GWD types: the GWD1, involved in the tagging of the glucan chains by C-6 phosphorylation, which is a prerequisite for subsequent C-3 phosphorylation by the second isoform, the GWD3/PWD (glucan, water dikinase 3/phosphoglucan, water dikinase) [37-39]. These enzymes seem to have evolved concomitantly with the appearance of starch deposition [36,40].

A fourth obligatory step in starch biosynthesis has been identified through genetic studies but is poorly understood in terms of the biochemical mechanism that mediates the effect. This step is the cleavage of α-1,6 linkages by isoamylase-type DBE [10,41]. The DBE are crucial for the generation of longer, clustered linear segments in the amylopectin molecule that can crystallize and increase the density of the polysaccharide [42]. Plants contain four DBE genes, three of which are classified as isoamylases on the basis of their sequence homologies and substrate specificities, and one pullulanase-type debranching enzyme [10,43].

While the steps leading to the synthesis of starch are common in most cereals, there are differences in the location and engagement of enzymes, depending on whether the synthesis is in leaf or endosperm (Figure 1).

**Figure 1.** Starch biosynthesis pathway in plants from Glc1P. The scheme indicates the involvement of different isoforms in cereal leaf (L) or endosperm (E). When not specified, the enzymes are dual localized in both compartments (adapted from [10] and [44]).
