**2. Carbohydrate production and nitrogen fixation**

Carbohydrates are produced in plants by photosynthesis. Zhu et al. (2010) reviewed photosynthesis in relation to improving crop yield. Agronomically, there has been little benefit in breeding for increased photosynthesis indicating that the relationship of photosynthesis to yield is still not well understood (Farquhar & Sharkey, 1982; Pessarakli, 2005). The relative growth rate of shoots was shown to be correlated to the soluble carbohydrate level in the plant, but shoot growth was also impacted by plant stress (Masle *et al*, 1990). One commonly studied plant stress in relation to carbohydrate production is drought stress. There is confusion regarding the regulation of carbohydrate synthesis when plants are under drought stress. Drought stress in addition to reducing shoot growth, increases root growth (Sharp & Davies, 1979).

Approximately 70 million tons of fixed nitrogen or about 50 % of the total nitrogen that enters the terrestrial ecosystem comes from biological nitrogen fixation (Brockwell *et al.*, 1995; Tate, 1995). The relationship of carbohydrate availability to photosynthesis, phloem sap supply and N2 fixation in legumes is complex and knowledge is incomplete (Udvardi & Day, 1997).

Extraction and Analysis of Inositols and Other Carbohydrates from Soybean Plant Tissues 423

Zeeman *et al*., 2010 reviewed the role of starches in plants. Starch breakdown commonly occurs when seeds germinate. Starch is also involved in malting (Halford *et al*., 2010). Glycogen, also a polymer of glucose, is the polysaccharide used by animals to store energy. Another important polysaccharide is cellulose. Cellulose is used as a structural molecule to add support to leaves, stems, and other parts of plants. Although cellulose can't be used as an energy source in most animals, it provides essential fiber in the diet. Cell wall polysaccharides vary with plant groups and can include cellulose, xyloglucan, arabinoxylan, and pectin. In plants they make up the primary biomass and contribute to fiber in the human diet. This area

The methods used for isolating carbohydrates depend on the carbohydrate type, matrix, and purpose or type of analysis. However, some extraction procedures are commonly used for isolating carbohydrates from other classes of compounds in plants and foods. As an example, foods are usually dried under vacuum to prevent thermal degradation, ground to a fine powder to enhance extraction efficiency, and then remove the fats using appropriate

A commonly used method for extracting low molecular weight carbohydrates from foods is to boil a sample with a 70-80% alcohol solution (Hall 2003, Asp 1993, Smith 1973.). Monosaccharides and oligosaccharides are soluble in alcohol solutions; however, most proteins, polysaccharides and dietary fiber are insoluble. The soluble components can then be separated from the insoluble components by filtering, soluble portion passes through the filter and the insoluble part retained by the filter. The two fractions can then be dried using lyophilization or nitrogen blow down techniques. In addition, monosaccharides and oligosaccharides and various other small molecules (e.g. organic acids, amino acids) may be present in the alcoholic extract. It is usually necessary to remove those components prior to carrying out a carbohydrate analysis, for example, with clarifying agents or by elution

Water extracts of many foods contain substances that are colored or produce turbidity, and may interfere with analyses of carbohydrates; as a result, clarifiers may be needed. The most commonly used clarifying agents are heavy metals (e.g. lead acetate) which form insoluble complexes with interfering substances that can't be removed by either filtration or centrifugation. Ion-exchange is another method for removing interfering components prior to analysis. Many monosaccharides and polysaccharides are polar non-charged molecules and can therefore be separated from charged molecules by passing samples through an ionexchange column. By using a combination of cationic and anionic resins it may be possible to remove most charged contaminants. Non-polar molecules can be removed by eluting through a column with a non-polar or hydrophobic stationary phase. Proteins, amino acids, organic acids, and hydrophobic compounds can be potentially removed from the

Before analysis of the carbohydrates, residual alcohol (or other organic solvents) can be removed, if necessary, from the solution by evaporating under nitrogen or under vacuum using a rotary evaporator. For aqueous solutions, the sample can be concentrated using

Solid phase extraction (SPE) has also been reported for the cleanup and quantification of sugars and organic acids in herbal dry extracts. A three step SPE sequence was used for the

has been reviewed by Scheller & Ulvskov, 2010; Fontes & Gilbert, 2010.

**5. Extraction and cleanup** 

through one or more ion-exchange resins.

carbohydrates in this manner prior to analyses.

solvent extraction.

lyophilization.

Carbohydrates are the main energy source for humans. Carbohydrates are classified according to the number of monomers they contain as monosaccharides (simple sugars), oligosaccharides, or polysaccharides. Carbohydrate metabolism in plants has been reviewed (Colowick & Kaplan, 1951; Ochoa & Stern, 1952; and Horecker & Mehler, 1955). Carbohydrate levels in soybean seed are highest at growth stage R 5.5, or when the seed is half-developed (Wilson, 2004). A significant portion of the carbohydrate produced by photosynthesis is respired in the plant roots. (Lambers *et al*., 1996).

#### **3. Simple sugars**

The most common simple sugars are glucose and fructose. Disaccharides consist of two covalently bound sugar molecules. Sucrose, for example, is a disaccharide consisting of glucose and fructose. Sugars have a role in energy, carbon transport molecules, hormonelike signaling factors, and as the source for building proteins, polysaccharides, oils and woody materials (Halford *et al*., 2010). Plant genotype and environment greatly affect the levels found in plants (Halford *et al*., 2010).
