Modern Biotechnology in Plant Breeding: Analysis of Glycoalkaloids in

Chapter 24

Modern Biotechnology in Plant Breeding: Analysis of Glycoalkaloids in Transgenic Potatoes Downloaded by UNIV OF CALIFORNIA SAN DIEGO on December 19, 2016 | http://pubs.acs.org Publication Date: August 13, 1996 | doi: 10.1021/bk-1996-0637.ch024

1

2

2

Karl-Heinz Engel , Werner K. Blaas , Barbara Gabriel , and Mathias Beckman 2

1

Lehrstuhl für Allgemeine Lebensmitteltechnologie, Technische Universität München, D-85350 Freising-Weihenstephan, Germany Bundesinstitut für Gesundheitlichen, Verbraucherschutz und Veterinärmedizin, Postfach 330013, D-14191 Berlin, Germany 2

Steroidal glycoalkaloids in potatoes are among the most prominent naturally occurring food toxicants. A mathematical correlation between tuber size and alkaloid content has been established, which allows the assessment of different potato varieties in terms of glycoalkaloid content by calculation for a normalized weight, independent from the tuber size analyzed. Potatoes modified by means of recombinant DNA techniques have been investigated with this method. Inhibition of amylose biosynthesis by anti-sense RNA expression had no effect on the glycoalkaloid content. However, insertion of an invertase gene from yeast caused a reduction of the concentrations of these critical food toxicants. These studies may serve as a basis for the application of the principle of "substantial equivalence" to the safety evaluation of transgenic potatoes. Modern biotechnology in plant breeding practices increasingly includes the application of recombinant DNA techniques (1). Genetically modified crops and their products have been commercialized or are about to enter the market (2-4). Genetic engineering in the development of foods raises a series of issues. In addition to basic ethical concerns, major emphasis has been placed on questions related to the release of genetically modified organisms into the environment (5). Due to the increasing transition from controlled field experiments to the placing of products on the market, food safety aspects are gaining importance (6). National and international organizations and authorities are in the process of developing strategies for the safety assessment of foods produced via recombinant DNA techniques (7, 8). The Organization for Economic Cooperation and Development (OECD) has elaborated the principle of "substantial equivalence", which is based upon comparison of the novel food to its traditional counterpart. If the new food or food component is found to be "substantially equivalent" to an existing food, it can be treated in the same manner with respect to safety (9). 0097-6156/%/0637-0249$15.00/0 © 1996 American Chemical Society

Takeoka et al.; Biotechnology for Improved Foods and Flavors ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on December 19, 2016 | http://pubs.acs.org Publication Date: August 13, 1996 | doi: 10.1021/bk-1996-0637.ch024

250

BIOTECHNOLOGY FOR IMPROVED FOODS AND FLAVORS

The World Health Organization (WHO) has developed a protocol on how to apply this principle to foods or food components from plants derived by modern biotechnology (10). The determination of "substantial equivalence" is based on a molecular characterization on the DNA level; it further includes the evaluation of agronomic traits, and it concludes with a chemical characterization of the new food or food component. The major focus of this chemical analysis is on naturally occurring toxicants and critical nutrients. One of the most prominent examples of naturally occurring food toxicants is steroidal glycoalkaloids in potatoes (77). Potatoes have been subjected to a wide spectrum of genetic modifications by means of recombinant DNA techniques (7214). This paper describes studies on the influence of genetic engineering on the amounts of naturally occurring glycoalkaloids which may serve as a basis for the application of the concept of "substantial equivalence" in the safety assessment of transgenic potatoes. Potato alkaloids. The cultivated potato (Solatium tuberosum L.) contains two major glycoalkaloids, a-chaconine and a-solanine. The two components both contain the C steroidal aglycone solanidine; they differ only in the sugar moieties included in the trisaccharide part. a-Solanine and a-chaconine form up to 95% of the glycoalkaloids present in potatoes. Data on occurrence, chemistry, analysis, and toxicology of the steroidal glycoalkaloids present in potatoes have been compre hensively reviewed (77, 75). A modified version of the procedures described by Bushway et al. (76) and Carman et al. (77) has been applied to investigate a-chaconine and a-solanine in potatoes: the compounds were extracted from potato tubers using an acidified mixture of chloroform and methanol. After evaporation of the organic solvents, the residue was redissolved in aqueous heptanesulfonic acid. This crude extract was purified using a C reversed phase cartridge. Separate quantification of the two alkaloids was finally achieved by HPLC. With this method, the glycoalkaloid content in experimentally grown and in commercially available potatoes was determined (18). Differences based on genetic variability as well as on geographic origin were observed. As examples, data obtained for varieties purchased from local grocery stores are presented in Figure 1. 27

18

Dependence of potato alkaloid content on tuber size. It has long been known that the alkaloid content of potatoes is inversely related to the size of the tubers (79, 20). The major portion of glycoalkaloids in potato tubers is located within the first mm from the outside surface, and decreases toward the center of the tubers (27). Therefore, glycoalkaloid concentrations are higher in small tubers due to the surface to mass ratio (22). This dependence of glycoalkaloid content on the tuber size reflected in the data presented in Figure 1 makes it difficult to compare analytical data. Because of the lack of linearity, it is not possible to take the average of tuber sizes investigated and to relate it to the average of the glycoalkaloid amounts determined. Therefore, the first goal was to establish a mathematical correlation between tuber size and glycoalkaloid content, thus establishing a basis for a comparison of glycoalkaloid data independent from the tuber size analyzed.



24.

ENGEL ET AL.

Analysis of Glycoalkaloids in Transgenic Potatoes 251

The investigation of a wide spectrum of different potato cultivars with tuber weights ranging from less than 10 g to more than 280 g revealed that the relationship between tuber size and alkaloid concentration is based on the equation shown in Figure 2a. The fact that this function is well suited to describe the correlation between tuber size and alkaloid content in potatoes becomes even more evident after transforming the equation as shown in Figure 2b. Plotting of the natural logarithm of the alkaloid content versus the natural logarithm of the tuber weight shows a linear relationship between these parameters. The coefficient "a" is a characteristic of each variety; it determines the position of the graphs on the y-axis and reflects the glycolkaloid concentration in the potato. Coefficient "b" determines the slope of the curves. Analyses of a broad spectrum of different potato varieties revealed that b averages -0.5 for a-solanine and -0.42 for a-chaconine, respectively (25). This correlation offers the possibility to calculate the alkaloid content for a certain size tuber on the basis of the results obtained from the analysis of any other tuber size. Glycoalkaloid contents (GA and GA ) of different tuber sizes (weights m and m ) are determined by the following equations: X

2

l

GA, GA

2

a + b • In m

2

a + b • In m

2

[1]

2

GA

=

2

GAj e

b (In m - In m ) 2

x

[2]

glycoalkaloids [mg/100g] 25 a

Hela

20 -»--—

50

i

I

i

100

150

200

i

•

250 300 tuber weight [g]

Figure 1- Glycoalkaloids determined in commercial potato varieties. Continued on next page



252


glycoalkaloids [mg/100g]

50

100

150

200


100

150

200


glycoalkaloids [mg/100g]

0

50

Figure 1. Continued



24. ENGEL ET AL.

Analysis of Glycoalkaloids in Transgenic Potatoes

glycoalkaloids [mg/100g] 30 a Karlena 25 -^a-solanine *a-chaconine 20 -

y

G A

•

15

=

e

a + b-lnx

a

=

+

b

l

n

m

e

GA = glycoalkaloids [mg/100g] m = tuber weight [g]

10 \

Ss

• ^*i

- v.

5

• •

t 50

n 0

•

g I 100

,

•

I 150

i 200

i 250 300 tuber weight [g]

In GA (GA = glycoalkaloids [mg/100g]) 4 Karlena

^^^^

GA

=

a

+

b

l

n

m

e

lnGA = a + b l n m

^^^^^ "

2 -•- a-solanine a=3,08 b=-0,50 a-chaconine a=3,52 b=-0,41 0| 0

l 1

i 2

i| 3

!' 4

i! 5

I 6 In m (m = tuber weight [g])

Figure 2. Mathematical correlation between glycoalkaloid content and potato tuber size: upper (a), lower (b).


253

254


In order to compare analytical data, a certain reference weight, e.g. 100 g, can be selected and glycoalkaloid concentrations (GA ) determined for any other tuber size (m ) can be normalized to this reference weight as follows: X

x


GA

100

=

QA, • e

b

( l n

1 0 0

"

l n

^

[3]

The application of this concept to the assessment of various conventionally bred potato varieties is shown in Figure 3. On the basis of the data normalized according to equation [3], the average glycoalkaloid content including standard deviation for the reference weight can be determined. This procedure allows the comparison of different cultivars in terms of alkaloid content independent from the tuber sizes analyzed. This is of special importance in the beginning of new breeding programs, when only small tubers are available (25). On the basis of these results, the investigation of glycoalkaloid contents in genetically engineered potatoes has been approached. Two types of potatoes modified via recombinant DNA techniques have been investigated. Genetically modified (anti-GBSS) potato. Potato starch consists of two components: amy lose (20%) and amylopectin (80%). Granule-bound starch synthase (GBSS) is the major enzyme involved in the formation of amy lose.

Figure 3. Glycoalkaloid contents in potato varieties normalized to a reference tuber weight of 100 g.



24.

ENGEL ET AL.


Expression of an anti-sense RNA has been applied to inhibit the biosynthesis of this enzyme, thus leading to potato starch consisting of more than 95% amylopectin (24). The potato variety Desiree has been transformed using Agrobacterium tumefaciens. Similar experiments to develop transgenic amylose-free potatoes have been described (25). In contrast to that approach, in the present study tuber specific expression has been achieved by using the patatin B33 promotor from Solanum tuberosum. The combined analytical/mathematical approach described above has been used to assess the glycoalkaloid content in genetically modified (GM) anti-GBSS potatoes. For example, data on material obtained from a field trial performed in Germany in 1994 are presented in Figure 4. For both, a-chaconine and a-solanine, quantitative determinations over a range of tuber sizes from 8 g to more than 250 g have been carried out (27). The curves plotted in Figure 4 show that there is no statistically significant difference in the concentrations of these toxicants between the parental line and the genetically modified potato. In order to quantitate the data, the normalization according to equation [3] has been carried out. As example, Figure 6 summarizes the results obtained for a-chaconine from field trials performed in 1993. On the basis of 11 parental and 13 anti-GBSS tuber sizes, respectively, average concentrations for a normalized weight of 100 g and the corresponding standard deviations have been calculated. There is no statistically significant difference in the concentrations of a-chaconine between the parental line and the potatoes modified by inhibition of the amylose biosynthesis. According to investigations of glycoalkaloids in amylose-free transgenic potatoes obtained by the use of a plant virus promoter, glycoalkaloid concentrations in transgenic clones were even lower than in the parental line (26). However, tuber sizes investigated have not been specified and only total glycoalkaloid concentrations (without differentiation of a-solanine and a-chaconine) have been determined in that study. Genetically modified (invertase) potato. In the second example investigated, the modification obtained by genetic engineering leads to the biosynthesis of a yeast invertase in potatoes. The corresponding gene from Saccharomyces cerevisiae has been fused to the coding region of the N-terminal signal peptide of the proteinase inhibitor II from Solanum tuberosum, thus targeting the enzyme to the cell wall (28) . Tuber specific expression has been achieved by using the patatin B33 promotor. The transformation has been carried out using Agrobacterium tumefaciens. Due to the activity of the inserted invertase, sucrose is cleaved into glucose and fructose. As sucrose is the major transport form for photosynthesis products in the plant, this causes changes in the source (leaves) / sink (tubers) interactions, which eventually lead to increased yield and/or increased tuber size (29) . The investigation of GM (invertase) potatoes grown under greenhouse conditions indicated a decrease of the glycoalkaloid concentrations in the genetically modified line compared to the parental control (Figure 5a). In order to verify these results, genetically modified material obtained from field trials has been investigated. Glycoalkaloid amounts have been determined in four independently transformed lines. Data determined for a-chaconine in one of these lines obtained


255

256


a-chaconine [mg/100g) 40 Desiree field trial 1994


35 30 a-chaconine:

25

•e- parental line * G M (anti-GBSS)

20 15 10 5 0

50

100

150

200


a-solanine [mg/100g] 20

> b

Desiree field trial 1994

15 -parental line a-solanine: ' — GM (anti-GBSS) 10

50

100

150

200


Figure 4. Glycoalkaloid content in genetically modified anti-GBSS potatoes (inhibition of amylose biosynthesis) and the corresponding untransformed control lines: upper (a), lower (b).


24.

257 Analysis of Glycoalkaloids in Transgenic Potatoes

ENGEL ET AL.

glycoalkaloids [mg/100g] 25


a

Desiree greenhouse 1994

-

20

-e-parental line • GM (invertase) -a-parental line a-chaconine: • GM (invertase)

a-solanine: 15 10 5 • •• o

1

•

n

•

%

•

g ••

0

i 50

0

100

150 tuber weight [g]

a-chaconine [mg/100g] 30

b

Desiree field trial 1994

25

•

20

-B- parental line a-chaconine:

15

• G M (invertase - line 3) B33-INV No. 33

10

° T

5 n

0

50

3

FT™

—~

"

.

"

.

I

I

I

100

150

200

~ ° I


Figure 5. Glycoalkaloid content determined in genetically modified potatoes expressing yeast derived invertase, grown in the greenhouse, upper (5a), and obtained from field trials, lower (5b).


258



from the field trial in 1994 are shown in Figure 5b. The corresponding graphs show the significant difference between the parental and the genetically modified line. Quantitation of the data revealed that amounts of a-chaconine calculated for a normalized weight of 100 g in the GM (invertase) potatoes are more than 30% lower than in the parental line (Figure 6). Similar reductions in glycoalkaloid concentrations have been determined for greenhouse and field trial experiments in 1993.

Desiree (field trial 1993)

[mg/ioog]

1 = parental line 2 = GM (anti-GBSS) 3 = GM (invertase) norm weight: 100 g

1

2

3

Figure 6. Average concentrations of a-chaconine (normalized to a reference tuber weight of 100 g) determined in untransformed potatoes var. Desiree (1) , and in transgenic lines modified by inhibition of amy lose biosynthesis (2) , and by expression of yeast derived invertase (3), respectively.

Conclusion. A mathematical correlation between tuber size and alkaloid content of potatoes has been established. On the basis of this correlation it is possible to compare potato varieties in terms of alkaloid content by calculation of the data for a normalized weight. This concept can be applied to assess conventionally bred potatoes as well as genetically engineered potatoes. Amylose inhibition by means of anti-sense RNA expression had no effect on the alkaloid content of the transgenic potatoes. This important result bears heavily on the safety assessment of this crop-gene combination.


24.

ENGEL ET AL.


259

Conversely, the expression of a yeast derived invertase in potatoes leads to a reduction of these naturally occurring toxicants. The elucidation of the biochemical reason for this reduction (dilution effect due to the increase of the tubers or metabolic interference with the sugar moieties needed for the biosynthesis of the glycoalkaloids) is part of our ongoing research.


Acknowledgments The authors thank Dr. A . G . Heyer (Institut fur genbiologische Forschung, Berlin, Germany) and Dr. H . Tiemann (Bundesanstalt fur Zuchtungsforschung in Gross Liisewitz, Germany) for providing potato samples.

References 1.

2.

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4. 5.

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7. 8. 9. 10.

11.

Willmitzer, L . New Developments in Plant Biotechnology; Lecture Publications, Vol. 20; Ernst Schering Research Foundation: Berlin, Germany, 1994. Redenbaugh, K . ; Hiatt, W . ; Martineau, B.; Emlay, D . In Genetically Modified Foods: Safety Aspects; Engel, K.-H., Takeoka, G., Teranishi, R. Eds.; A C S Symposium Series 605; American Chemical Society: Washington, D C , 1995, pp 72-87. Fuchs, R . L . ; Se, D . B . ; Rogers, S.G.; Hammond, B . G . ; Padgette, S.R. In The Biosafety Results of Field Tests of Genetically Modified Plants and Microorganisms; Jones, D . D . ; E d . ; Proc. of the 3rd International Symposium; The University of California: Oakland, C A , 1995. A C N F P (Advisory Committee on Novel Foods and Processes), Annual Report 1994, MAFF-Publications: London. The Biosafety Results of Field Tests of Genetically Modified Plants and Microorganisms; Jones, D . D . ; E d . ; Proc. of the 3rd International Symposium; The University of California: Oakland, C A , 1995. Genetically Modified Foods: Safety Aspects; Engel, K.-H., Takeoka, G., Teranishi, R., Eds.; A C S Symposium Series 605, American Chemical Society: Washington, D C , 1995, 243 pp. IFBC (International Food Biotechnology Council), Regulat. Toxicol. Pharmacol. 1990, 12, 1-196. Kessler, D . A . ; Taylor, M . R . ; Maryanski, J . H . ; Flamm, E.L.; Kahl, L . S . Science, 1993, 256, 1747-1832. Safety Evaluation of Foods Derived by Modern Biotechnology: Concepts and Principles, O E C D : Paris, 1993. Application of the Principles of Substantial Equivalence to the Safety Evaluation of Foods or Food ComponentsfromPlants Derived by Modern Biotechnology, Report of a WHO Workshop, WHO: Geneva, 1995. van Gelder, W . M . J . In Poisonous Plant Contamination of Edible Plants; Rizk, A.F.M., E d . ; C R C Press: Boca Raton, F L , 1991; pp 117-156.


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Modern Biotechnology in Plant Breeding: Analysis of Glycoalkaloids in

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