GLYCOLYTIC ENZYMES IN TISSUES OF SUGAR BEET
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GLYCOLYTIC ENZYMES IN TISSUES OF SUGAR BEET
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Sugarbeet is a temperate species whose taproots are used for sugar production. For maximum yield, however, it is essential to avoid sucrose degradation during cultivation and postharvest. Sucrose is cleaved by sucrolytic enzymes – sucrose synthase, acid invertase or alkaline invertase – and the products of these enzyme activities are catabolized by glycolysis to provide substrates for the TCA cycle, which provides substrates for the electron transfer chain. While sucrolytic enzymes have been studied in considerable detail, few studies have examined the activity of glycolytic enzymes. Determination of activities of glycolytic enzymes and measurement of flux of carbon through glycolysis is a necessary step to better understand the glycolytic pathway in sugarbeet roots and its contribution to sucrose loss. For determining enzymatic activities, sugarbeet roots grown in green house for 16 weeks were harvested, washed and stored for 0, 1, 2, 3, 4, 7, 10, 30, 60 and 100 days, with the first 10 days of storage at 10oC and subsequent storage at 4oC. Sugarbeet roots were evaluated for respiratory rates and then material was collected for the determination of enzyme activities. For carbon flux, roots were harvested and stored at 10oC in Experiment 1, and at 4, 10 and 20oC for Experiment 2 for 10 days. The lack of correlation between any individual glycolytic enzyme and root respiration rate during storage suggests that no single enzyme in the glycolytic pathway controls respiration rate. Canonic analysis identified four groups of enzymes that shared similarities in the manner in which their activities changed during storage. In experiments for carbon flux most of the radiolabel incorporated by root tissue remained as sucrose and was unmetabolized. Glucose and fructose concentrations were greater in roots disc incubated at 4oC than at 10 or 20oC. The increase of the incubation temperature provided relative changes in amount of labelled glucose, fructose and phosphoester glycolytic intermediates, suggesting that increased temperature increases the flux of carbon through early glycolytic enzymes to a greater extent than sucrolytic enzymes or late glycolytic enzymes.
TABLE OF CONTENTS
COVER PAGE
TITLE PAGE
APPROVAL PAGE
DEDICATION
ACKNOWELDGEMENT
ABSTRACT
CHAPTER ONE
INTRODUCTION
1.1 BACKGROUND OF THE STUDY
- AIM OF THE STUDY
- SCOPE OF THE STUDY
- SIGNIFICANCE OF THE STUDY
- PROJECT ORGANISATION
CHAPTER TWO
LITERATURE REVIEW
- OVERVIEW OF SUGAR BEET
- HISTORICAL BACKGROUND OF SUGAR BEETS
- DESCRIPTION OF GLYCOLYSIS
CHAPTER THREE
- MATERIAL AND METHODS
CHAPTER FOUR
RESULT ANALYSIS
- RESULTS AND DISCUSSION
CHAPTER FIVE
- CONCLUSION
- REFERENCES
CHAPTER ONE
1.0 INTRODUCTION
Glycolysis is the metabolic pathway that converts hexose sugars to pyruvate, releasing energy and generating substrates for the tricarboxylic acid cyle, respiration and the biosynthesis of biological compounds including amino acids, fatty acids, nucleic acids, phenolic compounds, and alkaloids. Overall, glycolysis catalyzes the following reaction:
glucose + 2 ADP + 2 Pi + 2 NAD+ → 2 pyruvate + 2 ATP + 2 NADH + 2 H+
Glycolysis is ubiquituous in nearly all organisms and serves as the primary pathway for carbohydrate catabolism. The oxidative pentose phosphate pathway (OPPP) provides an alternative pathway for carbohydrate catabolism. Glycolysis and the OPPP operate somewhat independently in plant cells, but interact through the common intermediates, glucose 6-phosphate, fructose 6- phosphate, and glyceraldehyde 3-phosphate (Bowsher et al., 2008). In most plants and plant organs, however, and in sugarbeet root in particular, glycolysis, is the predominant pathway for hexose catabolism (ap Rees, 1980; Wang & Barbour, 1961).
In plants, the number of participating enzymes is greater than the number of reactions since glycolysis can utilize two substrates, glucose and fructose, and three of the pathway’s reactions are catalyzed by two enzymes. Glycolysis requires 5 cofactors, ATP, ADP, NAD+, phosphate, and pyrophosphate. The pathway consumes energy in the form of ATP in its initial two steps, and produces 4 ATP in later reactions for an overall net energy yield of two ATP and two NADH molecules per hexose oxidized (Nelson & Cox, 2008).
Sucrose catabolism provides the substrates for glycolysis in sugarbeet root (Barbour & Wang, 1961). Sucrose catabolism is catalzyed by three enzymes in plants (Fig. 1): acid invertase (E.C. 3.2.1.26), alkaline invertase (E.C. 3.2.1.53), and sucrose synthase (E.C. 2.4.1.13, SuSy). In sugarbeet roots, all the three enzymes are found, although sucrose catabolism is thought to be largely catalyzed by SuSy (Echeverria & Gonzalez, 2003; Klotz & Finger, 2004). SuSy is a cytoplasmatic enzyme that catalyses the reaction of uridine 5’- diphosphate (UDP) with sucrose, generating fructose and UDP-glucose. The fructose formed in this reaction can readily be used as a substrate for glycolysis. UDP-glucose can also be utilized as a glycolytic substrate after its conversion to glucose 6-phosphate by the combined activities of UDP-glucose pyrophosphorylase (UDPase) and phosphoglucomutase (PGM) (Leigh et al., 1979). UDPase is a cytoplasmic enzyme that catalyzes the reversible reaction of UDP-glucose with pyrophosphate to form glucose 1-phosphate and UTP. PGM catalyzes the conversion of glucose 1-phosphate to glucose 6-phosphate. Although reversible, the equilibrium constant of PGM favors glucose 6- phosphate formation (Dennis et al., 1997).
Table 1. Glycolytic enzymes, Enzyme Commission (E.C.) numbers and abbreviations used.
Enzyme |
E.C. |
abbreviation |
Aldolase |
4.1.2.13 |
ALD |
ATP-dependent phosphofructokinase |
2.1.7.11 |
PFK |
Enolase |
4.2.1.11 |
ENO |
Fructokinase |
2.7.1.4 |
FK |
Glucose 6-phosphate isomerase |
5.3.1.9 |
G6PI |
Glyceraldehyde 3-phosphate dehydrogenase |
1.2.1.12 |
GAPDH |
Nonphosphorylating glyceraldehyde 3-phosphate dehydrogenase |
1.2.1.13 |
NGAPDH |
Hexokinase |
2.7.1.1 |
HK |
Phosphoglucose mutase |
5.4.2.2 |
PGM |
Phosphoglycerate kinase |
2.7.2.3 |
PGK |
Phosphoglycerate mutase |
2.7.5.3 |
PGlyM |
Phosphoenolpyruvate phosphatase |
3.1.3.60 |
PEPase |
Pyrophosphate-dependent phosphofructokinase |
2.7.1.90 |
PFP |
Pyruvate kinase |
2.7.1.40 |
PK |
Triose phosphate isomerase |
5.3.1.1 |
TPI |
UDP-glucose pyrophosphorylase |
2.7.7.9 |
UDPase |
Glycolysis starts with the phosphorylation of the hexose sugars, fructose and glucose, to glucose 6-phosphate and fructose 6-phosphate by the action of hexokinase (HK) and fructokinase (FK), respectively. Both enzymes catalyze irreversible reactions and consume ATP. HK and FK activities exhibit substrate specificities that allow independent regulation of glucose and fructose utilization (Renz & Stitt, 1993). Hexokinases are highly reactive with glucose and limitedly reactive with fructose; fructokinases react specifically with fructose (Claeyssen & Rivoal, 2007). In addition, HK can act as a hexose sensor and mediate changes in gene expression in response to carbohydrate status (Rolland et al., 2002). The glucose 6-phosphate formed by HK reaction is converted to fructose 6- phosphate by the action of glucose 6-phosphate isomerase (G6PI), a reversible enzyme with an equilibrium constant that slightly favors glucose 6-phosphate formation (Dennis et al., 1997).
Fructose 6-phosphate is converted to fructose 1,6-bisphosphate by the activities of ATP-dependent phosphofructokinase (PFK) and pyrophosphate- dependent phosphofructokinase (PFP). PFK, however, is primarily responsible for fructose 1,6-bisphosphate formation in most plant cells (Dennis & Blakely, 2000). PFK catalyses an irreversible reaction and is an important control point in the glycolytic pathway (Bowsher et al., 2008). In contrast, PFP, located exclusively in the cytosol, catalyzes a readily reversible reaction with an equilibrium constant that favors fructose 1,6 bisphosphate formation and operates near equilibrium in vivo (Plaxton, 1996; Dennis et al., 1997).
Aldolase (ALD) catalyzes an aldol cleavage of fructose 1,6-bisphosphate (F1,6BP) to form glyceraldehyde 3-phosphate and dyhydroxyacetone, two compounds that are interconverted by the action of triose phosphate isomerase (TPI). Both ALD and TPI catalyze reversible reactions with equilibrium constants that strongly favor F1,6BP and dihydroxyacetone formation, respectively (Dennis et al., 1997). Since the aldolase reaction equilibrium favors the reverse reaction, TPI has the important role of pulling the ALD reaction forward by keeping glyceraldehyde 3-phosphate concentrations low. Reactions in the lower part of the glycolytic pathway also consume glyceraldehyde 3-phosphate, and function to drive the ALD reaction forward (Bowsher et al., 2008).
Glyceraldehyde 3-phosphate is converted to 1,3-bisphosphoglycerate by glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The reaction links the oxidation of glyceraldehyde 3-phosphate to the reduction of NAD and requires inorganic phosphate (Bowsher et al., 2008). The GAPDH reaction is readily reversible with an equilibrium that favors glyceraldehyde 3-phosphate by about 10:1 over 1,3-bisphosphoglycerate (Dennis et al., 1997). The enzyme phosphoglycerate kinase (PGK) transfers a phosphate group from 1,3- bisphosphoglycerate to ADP. The reaction produces ATP and 3- phosphoglycerate and is the first of two ATP-generating reactions in glycolysis (Dennis et al., 1997; Nelson & Cox, 2008). While reversible, PGK reaction favors 3-phosphoglycerate formation. A bypass of the ATP-forming PGK reaction is catalyzed by nonphosphorylating glyceraldehyde 3-phosphate dehydrogenase (NGAPDH). NGAPDH catalyzes the oxidation of glyceraldehyde 3-phosphate to 3-phosphoglycerate with concommittal reduction of NADP+ to NADPH (Dennis et al., 1997).
Phosphoglycerate mutase (PGlyM) catalyzes the interconversion of 3- phosphoglycerate and 2-phosphoglycerate (Nelson & Cox, 2008). The 2- phosphoglycerate formed by PGlyM reaction is dehydrated by enolase (ENO) to produce the high-energy compound phosphoenolpyruvate (PEP) (Bowsher et al., 2008). Both PGlyM and ENO catalyze readily reversible reactions.
In the final reaction of glycolysis, PEP is converted to pyruvate by pyruvate kinase (PK). The phosphate attached to the 2-position of PEP has a high negative free energy of hydrolysis and is transferred to ADP with the resulting formation of ATP and pyruvate in an irreversible reaction (Bowsher et al., 2008). Conversion of PEP to pyruvate can also be catalyzed by phosphoenolpyruvate phosphatase (PEPase). PEPase cleaves the phosphate group from PEP without ATP formation. PEPase, however, is located in the vacuole and inhibited by inorganic phosphate and is thought to be inactive under normal cellular conditions (Dennis et al., 1997).
Control of glycolysis in plants is not well understood. In plants, PFK is thought to have a central role in the regulation of the pathway, with its activity regulated by glycolytic downstream products and intermediates. PFK activity is inhibited by low concentrations of PEP, moderate concentrations of 3- phosphoglycerate and 2-phosphoglycerate, and ATP. It is activated by inorganic phosphate and Mg+2. The major factor controlling PFK activity, however, is thought to be the ratio of PEP: Pi (Givan, 1999). The central role of PFK in regulating glycolytic flux, however, is uncertain. Several studies have demonstrated that alterations in PFK activitiy has little or no effect on glycolytic flux (Burrell et al., 1994, Thomas et al., 1997). Other studies have suggested that other glycolytic enzymes contribute to glycolytic control (Hatzfeld et al., 1989), and a growing body of evidence suggests that all enzymes of a pathway incrementally contribute to pathway regulation (Geigenberger et al., 2004).
Little is known about the activities of glycolytic enzymes or the regulation of glycolysis in sugarbeet root. In sugarbeet roots, hexokinase, fructokinase, phosphofructokinase, pyrophosphate dependent phosphofructokinase, aldolase, glucose 6-phosphate isomerase and pyruvate kinase activities have been determined in response to wounding and temperature (Klotz et al., 2006; Moorhead & Plaxton, 1988; Sakalo & Tyltu, 1997). The present study intends to determine the activity of glycolytic enzymes in sugarbeet roots throughout 100 days of storage. The purpose of this work is to provide basic information for understanding the activities of the enzymes involved in glycolysis, their relative activities, and the effect of storage on them.
1.1 BACKGROUND OF THE STUDY
Sugarbeet (Beta vulgaris L.) is produced throughout the world, especially in the USA, Russia, the European Union and Ukraine. Europe is the major producer sugar of sugarbeet and responsible for 75% of total beet sugar produced. The USA and Asia make up 19% of world production; South America and Africa account for the remainder (Draycott, 2006).
In 2007, the USA produced about 32 million tons of sugarbeet in 504 mil hectares (FAOSTAT, 2007). Production for the 2009/2101 crop has yet to be quantified, but the National Agricultural Statistics Service (NASS) forecasts that for 2009/10, sugarbeet will be harvested from 470 mil hectares, a 15.3 percent increase over that of 2008/09, and 31 million tons of sugarbeet will be produced. In contrast, in the USA sugar cane production is estimated at approximately 303 mil hectares and 4 million tons (Haley & Dohlman, 2009). These numbers demonstrate the importance of sugarbeet for sugar production in the USA.
In the USA, sugarbeet is cultivated in different regions including the Upper Midwest (Minnesota and North Dakota), the Far West (California, Idaho, Oregon, and Washington State), the Great Plains (Colorado, Nebraska, Montana, and Wyoming) and the Great Lakes (Michigan) (Harveson et al., 2009). The sugarbeet crop can be grown commercially in a wide variety of temperate climates, but is mostly grown at latitudes between 30 and 60oN (Draycott, 2006). In recent years, the development of tropical sugarbeet varieties has created an interest in cultivation in regions of tropical climates (Joshi et al., 2005).
Beta vulgaris L. is a member of the Chenopodiaceae family and is agriculturally important because of its ability to accumulate a large quantity of sugar in its storage root (Milford, 2006). The sugarbeet is a biennial plant, requiring vernalization (temperature near 6oC) and long-day conditions (photo- thermal induction) to induce flowering and seed production (Lewellen et al., 2009; Milford, 2006). In commercial beet production, the root is harvested after the first growing season. The crop needs a relatively long growing period, normally from 140-160 up to 200 days to produce a commercially viable root, which typically weighs 1-2 kg and contains 15-20% sucrose based on fresh weight (Jaggard & Qi, 2006). However, the growing period and consequently root yield and sucrose content are directly dependent on the production region (Went, 1954).
After harvest, sugarbeet roots are typically stored in large outdoor piles. Postharvest storage conditions and practices, however, vary substantially between production regions. In Mediterranean regions, including Southern Europe and North Africa, and in California, roots are processed shortly after harvest, usually within a few days, with harvest extending over two to five months to provide a constant supply of roots to processing factories. In Western Europe, sugarbeet roots are harvested until the first frost and roots are stored on-farm in piles called clamps for 3-6 weeks. In regions with short growing seasons and cold winters, e.g. those in the Northern USA, and parts of Russia, harvesting is delayed until just before the first frost, and roots are stored for up to 200 days prior to processing (Tungland et al., 1998). In the USA, roots are delivered to central piling stations where they are stored in piles 6-10 m high and 55-70 m wide and 400 m long. The piles are cooled by ambient winter air by natural or forced air ventilation. In these regions, roots may be stored up to 120 days prior to freezing or processing.
A major challenge for the sugarbeet industry is to preserve the sugar accumulated during postharvest storage. Sugarbeet roots lose, on average, 0.1% of their sucrose content per day in storage at 3oC, although losses can reach 1.8% of their sugar content per day at temperatures higher than 30oC (Wyse & Dexter, 1971). Given the duration of storage, it is common for 10 to 15% of the sucrose present at harvest to be lost during storage. Sucrose loss in storage is due to respiration, disease and conversion of sucrose to other carbohydrates including glucose, fructose, and raffinose. Respiration, however, is the principal cause of sucrose loss with estimates that 60 to 80% of the sucrose lost during storage is due to this process (Wyse & Dexter, 1971).
Respiration is the process by which compounds normally present in plant cells, such as starch, sugars, and organic acids are oxidatively degraded to carbon dioxide and water (Salveit, 2004; Siedow & Day, 2000). Concomitant with this catabolic reaction is the production of energy and intermediate molecules that are required to maintain the physiological integrity of living cells (Wyse, 1973; Salveit, 2004). In sugarbeet root, respiration utilizes sucrose as its principal substrate (Barbour & Wang, 1961) and generates the energy and substrates needed to maintain healthy tissue, heal wounds incurred during harvest and piling, and defend against pathogens (Wyse & Dexter, 1971; Wyse, 1973). Respiration, therefore, is essential for biological process, but economically detrimental, and limiting its impact on sucrose loss is a major goal for the sugarbeet industry.
Respiration involves a complex series of reactions and involves sucrolysis, glycolysis, the citric acid cycle (TCA), and electron transfer/oxidative phosphorylation (Campbell & Klotz, 2006). In sugarbeet root, respiration begins with cleavage of sucrose by sucrolytic enzymes which cleave sucrose to hexose sugars. Glycolytic enzymes degrade the hexose sugars to the three carbon organic acid, pyruvate and with a concomitant release of energy. Pyruvate enters the TCA cycle which degrades the pyruvate to carbon dioxide and generates NADH and FADH2 which are substrates for the electron transfer chain (Klotz et al., 2006; Salveit, 2004).
Many factors are known to affect postharvest sugarbeet respiration, including storage temperature, the extent of mechanical damage incurred during harvest and piling, and disease (Fugate & Campbell, 2009). The most important factor affecting the rate of respiration, however, is temperature. Respiration rates generally decline with decreasing temperature, with rates decreasing by approximately one half for each 10oC reduction in temperature (Wyse, 1973 and 1978). The optimum temperature range for stored sugarbeet is thought to be between 1.5 and 5oC. Injury caused by harvest and piling operations is substantial, with typical root injuries including root breakage, splitting, surface abrasions, cuts, loss of small fragments, and bruising. Root injury increases respiration rate within 24 hours and causes root respiration rate to be elevated throughout the duration of storage (Wyse & Peterson, 1979; Wiltshire & Cobb, 2000). Moreover, injury increases the incidence of storage disease since the two major storage diseases of sugarbeet roots, Botrytis and Penicillium, require a wound in the epidermis and exposure of internal tissues to establish infection (Mumford & Wyse, 1976). Storage diseases also increase root respiration rate, with the increase in root respiration rate proportional to the surface area of the infection (Mumford & Wyse, 1976).
While the environmental factors that affect sugarbeet root respiration rate are known, the internal factors regulating root respiration rate are unknown. In plants, respiration rate is regulated by substrate availability, total respiratory activity, or energy status of the cell (Klotz et al., 2008; Shugaev & Bukhov, 1997). In sugarbeet root, the mechanism by which respiration rate is controlled has not been established. Studies, however, have demonstrated that root respiration rate is not associated with total respiratory activity or energy status of the cell. The lack of association between root respiration rate and total respiratory activity and cellular energy status suggests that respiration is regulated by the availability of respiratory substrates (Klotz et al., 2008).
Understanding the processes involved in the generation of substrates for respiration is, therefore, extremely important to understanding postharvest sucrose loss in sugar beet roots. The metabolic reactions involved in sucrose catabolism are well known. Sucrose is cleaved by one of three sucrolytic enzymes – sucrose synthase, acid invertase or alkaline invertase – and the products of these enzyme activities are catabolized by glycolysis to provide substrates for the TCA cycle, which provides substrates for the electron tranfer chain (Siedow & Day, 2000; Klotz & Finger, 2004). While sucrolytic enzymes have been studied in considerable detail, few studies have examined the activity of glycolytic enzymes and the flux of carbon substrates through this pathway. The paucity of studies dealing with the glycolytic pathway is surprising considering its central role in plant metabolism and its ability to limit respiration and anabolic pathways by restricting the availability of substrates for these processes.
In sugarbeet root, little information of glycolytic enzyme activities and their changes during storage is known. To date, hexokinase, fructokinase, phosphofructokinase and pyruvate kinase activities have been determined in response to wounding (Klotz et al., 2006), hexokinase, phosphofructokinase and pyruvate kinase were determined in dormant and sliced and aged roots (Moorhead & Plaxton, 1988), and hexokinase, fructokinase, and glucose 6- phosphate isomerase activities have been determined after storage at unusually high temperatures (Sakalo & Tyltu, 1997). These studies determined the activities of the glycolytic enzymes that have traditionally been considered regulatory of the pathway. Current research, however, has questioned the regulatory importance of those reactions and suggested that other enzymes in the pathway may contribute to the regulation of glycolysis. Thus, determination of activity of all glycolytic enzymes and measurement of the flux of carbon through glycolysis is a necessary step to better understand the glycolytic pathway in sugarbeet roots.
1.2 AIM OF THE STUDY
The aim of this work was to provide basic information for understanding the activities of the enzymes involved in glycolysis, their relative activities, and the effect of storage on sugar beet.
1.3 SCOPE OF THE STUDY
Glycolysis is ubiquituous in nearly all organisms and serves as the primary pathway for carbohydrate catabolism. The oxidative pentose phosphate pathway (OPPP) provides an alternative pathway for carbohydrate catabolism. Glycolysis and the OPPP operate somewhat independently in plant cells, but interact through common intermediates. In most plants and plant organs, however, and in sugarbeet root in particular, glycolysis, is the predominant pathway for hexose catabolism. Control of glycolysis in plants is not well understood. Little is known about the activities of glycolytic enzymes or the regulation of glycolysis in sugarbeet root. The present study determined the activity of glycolytic enzymes in sugarbeet roots throughout 100 days of storage.
Sugarbeet hybrid VDH66156 was greenhouse grown for 16 weeks, when roots were harvested, gently hand washed, and kept at 10oC and 90 ± 5% relative humidity for 10 days, then incubated at 4oC, 90 ± 5% relative humidity for up to 100 days. Respiration rate of individual roots was determined at 10oC after 0, 1, 2, 3, 4, 7, 10, 30, 60 and 100 days of storage, then tissue samples were flash frozen in liquid nitrogen, lyophilized, ground to a fine powder, and stored at −80oC until analysis of enzymatic activity. Respiration rate was nearly 23 mg CO2 kg-1 h-1 after harvest, but declined 65% after one day of storage. A second decline of 55%, occurred during the first week in storage to a rate of 3.6 mg CO2 kg-1 h-1, after which respiration rate was practically. The high activities observed for TPI, UDPase, G6PI and PGM suggest that the reactions catalyzed by these enzymes occur readily. The high activities observed for G6PI and PGM suggest that the hexose phosphates, fructose 6- phosphate, glucose 6-phosphate and glucose 1-phosphate, are readily.
1.4 SIGNIFICANCE OF THE STUDY
This work helps student involved to become conversant with different enzymes in tissues of sugar beet.
1.5 PROJECT ORGANISATION
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