Starch synthesis and its manipulation
Starch is a hugely important substance, both to plants and to humans. For plants, it is a compact form in which excess sugars produced during photosynthesis can be stored for later use. For humans, it is a vital component of our diet, and an important commercial product. The UK alone consumes approximately 880,000 tonnes of starch annually, three-quarters of which is eaten, while the rest is destined for industry. Starch and its derivatives are already widely employed in the manufacture of paper, textiles and adhesives, and due to their biodegradable and renewable nature they are increasingly being considered as an environmentally-friendly alternative to the use of synthetic additives in many other products, including plastics, detergents, pharmaceutical tablets, pesticides, cosmetics and even oil-drilling fluids.
All of this starch is derived from the grains and tubers of plants; in the UK, the main sources are maize, wheat and potato. Genetic manipulation of these plants has the potential to revolutionise the production of starch, increasing the yield of food crops, or allowing the manufacture of starches whose physical and chemical properties are tailored to meet specific industrial needs. In this essay, I will examine the progress that science has made towards understanding the biochemical processes by which plants synthesise starch, and manipulating these processes for the benefit of mankind.
The structure and synthesis of starchStarch, a complex carbohydrate, is a polymer of glucose molecules. It occurs in two main forms: amylose, consisting of predominantly linear chains of glucose monomers linked by 1,4-glycosidic bonds, and amylopectin, in which the chains are branched by the addition of 1,6-glycosidic bonds. Amylose comprises between 11 and 37% of the starch found in plants (depending upon the species and the site of storage); the rest is amylopectin.
The synthesis of starch in plant cells begins with the enzyme ADP-glucose pyrophosphorylase (AGPase), which catalyses the reaction of glucose-1-phosphate with ATP to form ADP-glucose (liberating pyrophosphate). The ADP-glucose is then used a substrate by starch synthase enzymes, which add glucose units to the end of a growing polymer chain to build up a starch molecule (releasing the ADP in the process). Branches in the chain are introduced by starch branching enzymes (SBEs), which hydrolyse 1,4-glycosidic bonds, and in their place, create 1,6 bonds with other glucose units.
Although the pathway of starch synthesis appears relatively simple, it is complicated by the fact that the enzymes involved come in various different forms, which differ in their behaviour and in the parts of a plant in which they are active. Further complexity is created by the presence of de-branching enzymes (DBEs), which hydrolyse 1,6-glycosidic bonds and break apart branches in the polymer chains. Although these are traditionally regarded as catalysts of starch breakdown, it appears that they also play an important role in starch synthesis. Evidence for this comes from the 'sugary' mutants of maize, rice and sorghum, which are deficient in a particular de-branching enzyme, in which starch granules are degraded as they form and replaced with an alternative polymer, phytoglycogen.
Increasing starch yield
One of the simplest and most obvious modifications that could be made to starch synthesis would simply be to increase the rate at which it occurred, encouraging crop plants to divert as much of their energy as possible into the production of starch. Improving the nutritional yield of food crops is vital if a growing world population is to be kept adequately fed without increasing the pressure on land use, and starch - which accounts for a large proportion of the energy content of many staple foods (including rice, potatoes and cereals) - is an obvious target for such modification. Enhancing the starch content of crops would also allow starch to be produced more cheaply for industrial use, enabling it to compete more effectively with non-biological alternative products.
The rate of starch synthesis is largely determined by the activity of AGPase, the first enzyme in the pathway. The form of this enzyme found in leaves is activated by 3-phosphoglyceric acid (3-PGA), a product of photosynthesis, and inhibited by inorganic phosphate (Pi), which accumulates when the rate of photosynthesis declines. This causes starch production to be increased at times when photosynthesis is proceeding rapidly and there are surplus sugars to be stored, and decreased during leaner times. An attempt was made to increase the yield of starch in potatoes by genetically replacing the normal AGPase with a bacterial version of the enzyme, which is not sensitive to 3-PGA and Pi, thus bypassing the regulatory mechanism. This experiment gave conflicting results, depending upon the potato variety used. In the Russell Burbank potato variety, the modification produced a 30% increase in starch content, but in the Prairie variety there was no such change. There may be a limit to the amount of starch that a potato can accumulate, which the Prairie variety had already reached, or the difference may result from pleiotropic effects of the modified AGPase on other enzymes in the potatoes. An alternative means of enhancing the activity of AGPase is to induce random mutations in the wild-type enzyme, so as to generate variants that are more readily activated by 3-PGA and less readily inhibited by Pi, leading to greater starch production.
AGPase, like most of the enzymes involved in starch synthesis, naturally occurs in multiple forms within a plant - the AGPase found in the endosperm of seeds (the main source from which starch is commercially extracted) is different from the AGPase used by leaves. The advantage of this is that genetic modifications could potentially be targeted in such a way as to increase starch production in the specific part of the plant where it is most useful (for example, in the grain of cereals).
Instead of modifying the AGPase enzyme itself, some researchers have investigated ways to enhance the rate of starch synthesis by increasing the availability of the enzyme's substrates. Increasing the levels of a 'plastidic ATP–ADP transporter', a protein involved in supplying ATP for the reaction, was found to increase the accumulation of starch in potatoes. The levels of several other proteins, including an adenylate transporter and a pyrophosphatase enzyme, are also known to influence the quantity of starch produced by a plant, making them possible targets for future genetic engineering to improve yields.
Amylose and amylopectin
For many commercial uses it is desirable to alter the proportions of amylose and amylopectin found in starch. High-amylose starches are useful in confectionery (because they thicken rapidly), in fried snacks (because they resist the penetration of cooking oil), and in photographic film (because of their toughness and transparency). It has also been suggested that the nutritional properties of bread can be improved by the use of flour high in amylose. Amylopectin is preferred in paper-making and adhesives (because its branched chains give it greater binding power), and in frozen food (because it enhances stability and shelf-life).
In both maize and wheat, naturally-occurring mutants are known whose starch consists entirely of amylopectin, with no amylose whatsoever. These so-called 'waxy' varieties result from damage to the genes coding for granule-bound starch synthase (GBSS), an enzyme specifically involved in the production of amylose. (The fact that amylopectin synthesis is not disrupted in these mutants illustrates that the pathway leading to its production involves different starch synthase enzymes.) In wheat there are three genes coding for isoforms of GBSS, and the loss of only one or two of these genes results in a partial reduction in amylose content. Waxy maize has been grown commercially for nearly a century, and is now widely-used to produce amylose-free starch for use in industry and as a food additive. However, current waxy wheat varieties are unsuitable for widespread cultivation.
Mutations in other starch synthases have also been identified (in peas, maize and the alga Chlamydomonas) that reduce the synthesis of amylopectin, increasing the amylose content of the starch produced. Mutations in starch synthase genes (or the suppression of these genes by antisense techniques) can also alter the branching pattern of amylopectin, altering the average length of the polymer chains. Genetic manipulation of starch synthases might, therefore, eventually provide a means of modifying the physical and chemical properties of starch in useful ways.
Although the activity of different starch synthases and SBEs is clearly important in determining the balance between amylose and amylopectin synthesis, it may not be the only factor involved. Studies in rice show that the temperature at which a plant develops can also influence the proportions of amylose and amylopectin produced, by affecting the activity of different starch synthase enzymes. There is also evidence that altering the rate at which starch is produced can affect the amylose/amylopectin ratio; mutations in pea plants that reduce the total rate of starch synthesis also increase the proportion of amylopectin present. One possible explanation is that the rate of amylopectin synthesis is normally limited by the available concentration of the necessary SBE, so at lower rates of production a greater proportion of the starch can be converted into amylopectin. This raises the worrying possibility that attempts to improve yield in plants might result in starch with increased amylose content, which would be undesirable for many commercial purposes.
Before use in many industrial processes, starch must be chemically modified - for example, by phosphorylation - in order to alter its physical properties (such as viscosity and gel-forming ability), and to prevent it from crystallising. If such modification could be carried out by the plant that produced the starch, it would reduce the need for expensive and environmentally-damaging chemical treatments.
Natural starch from many plant species does contain small amounts of covalently-bound phosphate (which may help to stabilise the physical structure of starch grains and play a role in starch breakdown). A crucial enzyme responsible for phosphorylating starch (an alpha-glucan water dikinase) has recently been identified in potatoes (potato starch is particularly rich in phosphate), and in future it is hoped that genetic modification of plants may permit the production of high-phosphate starch for use in industry. However, the biochemical processes involved are still poorly understood, and the extent of phosphorylation may depend upon the composition of the starch as well as the concentration of the necessary enzymes. There is evidence that starch with longer polymer chains tends to contain higher levels of phosphate because the longer chains provide a better substrate for the phosphorylating enzyme. This would complicate any attempt to increase the phosphate content of natural starch by genetic modification.
Several enzymes have been identified that can influence the amount or type of starch that a plant produces, and these may provide useful targets for future bioengineering. Alterations to the structure of the crucial enzyme AGPase have been shown in experiments to improve the quantity of starch produced by plants such as potatoes, and there are other enzymes that also influence starch yield. Genetic manipulation of starch synthase genes allow the proportions of amylose and amylopectin in starch to be altered, producing starches tailored for different industrial purposes. One naturally-occurring 'genetic modification', the waxy mutation that prevents maize from synthesising amylose, is already widely used to produce commercial starch consisting purely of amylopectin. A crucial enzyme involved in phosphorylating starch has been identified, and may be a future target for bioengineering to create plants that synthesise starch phosphate for industrial use.
However, despite the progress that has already been made in analysing and manipulating the mechanism of starch synthesis in plants, the complex interactions between the many enzymes involved are not yet fully understood, and attempts to manipulate starch synthesis can have unintentional side effects. For example, increasing the yield of starch in a plant may affect the proportions of amylose and amylopectin that it contains. The rugosus mutation in pea plants, whose primary effect is to reduce starch production (due to a defective SBE), also results in wrinkled seeds that contain abnormal quantities of lipid and storage proteins. If any attempt were made to improve the starch yield of food crops, it would be important to make sure that other nutritional qualities were not adversely affected.
One recurring theme when investigating the pathway of starch synthesis is that most of the enzymes involved come in multiple forms, which differ in their physical and chemical properties and in the type of starch that they produce. Although this increases the complexity of the process, and makes it more difficult to interpret the role of the different enzymes, it also increases the range of possibilities for bioengineering. By targeting specific forms of an enzyme (such as GBSS), it is possible to exercise control over the types of starch molecule that are produced.
There are also biochemical differences in starch synthesis between different species of crop plant. An example of this is in the form of AGPase that is found in seed endosperm. In potatoes and maize, AGPase in the endosperm is regulated by 3-PGA and Pi, much like it is in leaves, but in barley and wheat the AGPase enzyme found in the endosperm does not appear to be sensitive to such regulation. Such differences would need to be taken into account when engineering plants so as to modify starch synthesis - techniques learned with one plant species will not necessarily apply to another.
In addition to the chemical steps in starch synthesis that are discussed in this essay, there are also physical aspects to the process - such as the way in which starch molecules are organised into granules - into which further research needs to be done before our knowledge of starch synthesis is complete. Nonetheless, rapid advances are currently being made in our understanding of the way in which plants produce starch, and there is hope that technology will soon be capable of enhancing this vital biological process.
Garth Entwistle, Sheila Bachelor, Elaine Booth and Kerr Walker (1998), Economics of starch production in the UK. Industrial Crops and Products, Volume 7, Issues 2-3, January 1998, Pages 175-186.
Casey J. Slattery, I. Halil Kavakli and Thomas W. Okita (2000), Engineering starch for increased quantity and quality. Trends in Plant Science, Volume 5, Issue 7, 1 July 2000, Pages 291-298.
Martha G James, Kay Denyer, and Alan M Myers (2003), Starch synthesis in the cereal endosperm. Current Opinion in Plant Biology, Volume 6, Issue 3, June 2003, Pages 215-222.
Alison M. Smith (1999), Making Starch. Current Opinion In Plant Biology, Volume 2, Pages 223-229, 1999.
Cathie Martin and Alison M. Smith (1995), Starch Biosynthesis. The Plant Cell, Volume 7, Pages 971-985, July 1995.
A. Åkerberg, H. Liljeberg and I. Björck (1998), Effects of Amylose/Amylopectin Ratio and Baking Conditions on Resistant Starch Formation and Glycaemic Indices. Journal of Cereal Science, Volume 28, Issue 1, July 1998, Pages 71-80.
R. A. Graybosch (1998), Waxy wheats: Origin, properties, and prospects. Trends in Food Science & Technology, Volume 9, Issue 4, April 1998, Pages 135-142.
Takayuki Umemoto, Yasunori Nakamura and Norimitsu Ishikura (1995), Activity of starch synthase and the amylose content in rice endosperm. Phytochemistry, Volume 40, Number 6, Pages 1613-1616, 1995.
Andreas Blennow, Tom H. Nielsena, Lone Baunsgaarda, René Mikkelsena and Søren B. Engelsen (2002), Starch phosphorylation: a new front line in starch research. Trends in Plant Science, Volume 7, Issue 10 , 1 October 2002, Pages 445-450.
This was originally written as a university biology essay