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2.3. GM Potato
Potatoes are genetically modified to achieve a changed starch composition such
as an enhanced amylopektin fraction, or resistance to insects. Potatoes are known to
contain the inherent plant toxins and antinutrients solanine and other glykoalkaloids, but
furthermore, several proteaseinhibitors or phenols (e.g. chlorogenic acid) are also
present. Submitting a modified potato would need to show that the genetic modification
had not, for instance, inadvertently increased alkaloid levels.
Genetically modified starch potatoes with altered starch composition were
analysed for glycoalkaloid and chlorogenic acid content. The amount of glycoalkaloids
can vary for different reasons, for example cultivar differences, yield, stage of tissue
development and different types of stress. The genetic modification is not supposed to
influence the content of these substances, and this was verified in the analyses executed:
from the statistical analysis it is concluded that the amount of chlorogenic acid is not
affected by the genetic modification (Table 1).
Table 1. Content of chlorogenic acid, solanine, chaconine, total glycoalkaloids and
trypsin-inhibitors in modified potatoes
There were no significant differences in glycoalkaloid levels between different
clones, but in a later reply-letter it was stated that the contents of glycoalkaloids are
significantly smaller in the transformed potato than in the recipient variety (Table 1).
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In summary, it was stated that there are no increased contents of any of the
inherent plant toxins and antinutritional substances examined.
Nutritional and toxicological consequences of a genetic modification of potato in
respect to the amylopectin content were investigated. The analysis on inherent plant
toxins and antinutritional factors showed that the genetically modification did not
change the total glycoalkaloid content in the potato, but the composition of the
individual alkaloids could have been changed. Analyses of feed for a subchronic oral
toxicity trial with rats with modified and control potatoes did not reveal noticeable
differences between the total alkaloids of the different diets (Table 1).
Furthermore, protease-inhibitors, especially trypsin-inhibitors, were specified in
the analyses of the feed for the subchronic trial (Table 1). It is only mentioned that the
in vitro trypsin-inhibitor activity in unheated potatoes is considerably lower compared
to that in toasted soybeans, which are also used in livestock feed.
Other inherent plant toxins and antinutrients such as phenolic compounds and
coumarins are not considered relevant in these documents and therefore they were not
tested. For detailed data on the contents of chlorogenic acid, solanine, chaconine, total
glycoalkaloids and trypsin inhibitors in different modified potatoes (Table 1)
Also, transgenic potato plants containing genes encoding for different classes of
potentially insecticidal plant proteins, namely lectins,
-amylase inhibitors and
chitinases, have been investigated. High levels of expression of the foreign proteins,
which act as inherent plant toxins and antinutrients, were readily achieved throughout
the leaf and stem tissue, and in the tubers. The expression of the lectin in transgenic
potato plants caused significant detrimental effects to larvae.
Recently, data were published about genetically modified potato lines expressing
the gene of snowdrop bulb lectin (GNA). In preliminary rat feeding trials the transgenic
potatoes induced significant changes in the weights of some or most of the rats’ vital
organs, especially immune organs. Analysis shows that the contents of some of the
constituents of major nutritional importance in these genetically modified potatoes are
significantly different from those of their respective parent lines: protein and starch
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and/or glucose contents were different, similar findings were made for constituents such
as lectin and trypsin- and chymotrypsin-inhibitors. The changes in major components in
potato tubers after GNA-gene insertion and decreased foliar glycoalkaloid content in
various lines of genetically modified potatoes may have occurred by mechanisms such
as gene silencing, suppression and/or somaclonal variation as a result of gene insertion.
Results have been discussed controversially, and an audit committee was of the opinion
that the existing data do not support any suggestion that the consumption by rats of
transgenic potatoes expressing GNA has an effect on growth, organ development or
immune function. In any case, the results show that there is a lack of equivalence in
composition between parental and modified potatoes which affects metabolic
consequences of feeding.
2.3.1. Reduction of Cholesterol and Glycoalkaloid Levels in Transgenic
Potato Plants by Overexpression of a Type 1 Sterol Methyltransferase cDNA
Glycoalkaloids are a family of steroidal toxic secondary metabolites present in
plants of the Solanaceae family. In cultivated potato (Solanum tuberosum) the main
glycoalkaloids, -chaconine and -solanine, are triglycosylated products of the same
aglycone, solanidine, but they differ in their sugar moieties. The highest glycoalkaloid
level in potato plants is found in flowers and sprouts, followed by the leaves, and the
lowest amounts are detected in stems and tubers. The amount of glycoalkaloids
increases upon wounding and light exposure, something that may render tubers
unsuitable for human consumption. Mild clinical symptoms of glycoalkaloid poisoning
include abdominal pain, vomiting, and diarrhea, and an upper safe limit in tubers of
200 mg total glycoalkaloids (TGA) kg
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fresh weight has been recommended by
leading authorities. However, this upper limit is close to levels found in tubers destined
for human consumption, and efforts should be made to keep TGA levels low when
introducing new varieties on the market.
The biosynthesis of glycoalkaloids in potato is currently not fully understood.
Solanidine has been proposed to be synthesized from the key precursor in plant sterol
synthesis, cycloartenol, in a biosynthetic route including cholesterol, a sterol lacking
alkylations at the C-24 position in the side chain. Cholesterol is in most plant species
only a minor sterol, but is present at relatively high levels, approximately 15% to 20% of
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total sterols, in Solanaceous plants such as potato and tobacco (Nicotiana tabacum). One
of the final reactions in the synthesis of glycoalkaloids is the glucosylation or
galactosylation of solanidine to yield -chaconine or -solanine, respectively. A cDNA
encoding the solanidine glucosyltransferase (SGT) enzyme has been cloned. The SGT
mRNA increased after wounding, in line with previous measurements of woundinduced SGT activity and glycoalkaloid levels. However, the galactosylation of
solanidine is likely catalyzed by a separate enzyme. The final glycosylation steps
leading to -chaconine and -solanine have not been characterized.
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Figure 1.
Schematic presentation of proposed sterol and glycoalkaloid biosynthesis
pathways in potato plants. Dashed arrows indicate more than one enzymatic step. The
methylation steps catalyzed by SMT1 and SMT2 are indicated.
Cycloartenol metabolism leads also to the synthesis of other plant sterols. Plant
plasma membranes commonly contain a mixture of sterols, the main ones being the 24ethyl sterols sitosterol and stigmasterol, which together often constitute more than 70%
of total sterols. The alkylations of the sterol side chain are performed by the sequential
action of two distinct S-adenosyl-L-Met:sterol C24-methyltransferases, SMT type
1 (SMT1) and type 2 (SMT2). In the first step, cycloartenol is methylated to 24methylene cycloartanol by the enzymatic action of SMT1, whereas in the second
alkylation step, 24-methylene lophenol is methylated to 24-ethylidene lophenol by
action of SMT2. Several lines of evidence suggest a key role of the SMTs in the
synthesis of sterols and brassinosteroids, sterol-derived plant growth hormones.
Scientists were
reported a build-up of cycloartenol in ageing potato discs and
suggested that the activity of cycloartenol-C24-methyltransferase (SMT1) was limiting
in sterol synthesis. In line with this, transgenic plants overexpressing 3-hydroxy-3methylglutaryl CoA reductase, an early-acting enzyme in sterol synthesis, displayed up
to 60-fold higher levels of cycloartenol but much lower increases of 24-methylene
cycloartanol and its further metabolites. Furthermore, overexpression of SMT1 in
transgenic tobacco plants increased 24-methylated sterols at the expense of cholesterol,
whereas overexpression of SMT2 mainly increased the 24-ethyl sterols, this also at the
expense of cholesterol. In the SMT2 transformants, growth was reduced, presumably
due to a reduction of sterols needed in brassinosteroid synthesis. On the basis of the
analysis of transgenic Arabidopsis plants over- or underexpressing SMT2, Scientists
proposed a crucial role of SMT2 in balancing the ratio of campesterol to sitosterol to fit
both growth requirements and membrane integrity.
Because overexpression of either SMT1 or SMT2 in transgenic tobacco plants
leads to reduced levels of cholesterol, presumably due to an increased channeling of
cycloartenol into alkylated sterols, we reasoned that this might enable the precursor role
of cholesterol in TGA synthesis to be experimentally tested in transgenic potato plants.
Considering the negative effects of SMT2 overexpression on plant growth, we chose a
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SMT1 for this purpose. We here report on an altered sterol composition and a reduced
TGA level in such SMT1-overexpressing potato plants.
2.3.2. Introduction of Genetic Engineering in Potato Breeding
Three groups of actors are directly engaged in the introduction of genetic
engineering in potato breeding: public research institutes, biotechnology firms, and
potato breeding companies
The potato breeding companies, the third group of actors, are currently going
through a process of restructuring. First of all, there is a concentration of companies
under way, through mergers and takeovers. Second, potato breeders have expanded
breeding research activities, as potato breeding research at public institutes has been
restructured. Third, the rise of genetic engineering has encouraged companies to
increase investments in biotechnology research, either in-house or as contract research.
All companies feel the need to stay in touch with the latest research findings. Fourth,
pressure has increased on breeding companies to come up with varieties that have better
pest and disease resistance, and thus need less pesticides. As a result of the restructuring
process, the potato breeding companies will become stronger and more influential in the
potato product chain as a whole.
2.3.3. Applications of Transgenic Potatoes
Two kinds of transgenic potatoes can be distinguished: those with better
resistance to pests and diseases, and those with improved characteristics for storage and
processing.
All important potato pests and diseases are subject to genetic engineering
research, including diseases and loss caused by nematodes, viruses, fungi, bacteria,
insects, and herbicides. Although several of these pests and diseases are not a major
threat to potato cultivation , they are important because of the large export of seed
potatoes
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The first genetically modified potatoes were virus resistant potatoes. Transgenic
potatoes with resistance to Potato Virus X (PVX) will be ready for commercial
introduction within a few years. Currently these potatoes are subject to cultivation tests.
Genetic engineering research on resistance to nematodes and fungi - the two most
important threats to the potato crop - is only in its infancy. Commercial introduction of
transgenic potatoes with improved resistance to nematodes or fungi is not expected until
the end of the century.
Transgenic potatoes with improved storage and processing characteristics have
already been developed, and are now subject to cultivation tests. In one kind of
transgenic potato, the starch content has been changed. This amylose-free potato may
make industrial starch processing more efficient. Another kind of transgenic potato has
been made less vulnerable to bruising in order to reduce the loss of raw material during
storage, transport, and processing.
Research on transgenic potatoes is directed at
improving cold resistance. Potatoes with better cold resistance can be stored at lower
temperatures, and therefore need less chemicals for restraining sprout growth. In many
countries, genetic engineering research is directed at enhancing the starch content.
2.3.4. Economic Aspects of Transgenic Potatoes
Because both the efficacy and the price of transgenic potatoes are still unknown,
it is difficult to make a quantitative assessment of their economic impact. However, it is
already evident that genetic engineering research requires potato breeders to increase
their investments. Whether and when these investments will yield profits is still very
uncertain. The necessity of making this kind of investment furthers the process of
concentration among breeding companies. Return on investment in genetic engineering
research is also dependent on the system of intellectual property protection.. National
and international systems that pertain to plant breeding rights are currently under
discussion.
For potato farmers, the introduction of transgenic potatoes with improved
disease resistance may lead to a shift in variable costs: lower costs for pesticides but
higher costs for starting material. Transgenic potatoes with improved storage or
processing characteristics may earn higher prices for potato farmers, while here too, the
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starting material may be more expensive. However, the economic impact can only be
stated in hypothetical terms.
The introduction of transgenic potatoes will reinforce the vertical integration in
the potato chain. Transgenic potato varieties will only be grown under approval from
the customer (trading firm, processing company, retailer or consumer). This approval
will only be given if the transgenic variety has some evident qualitative improvement
when compared to non-modified varieties. Qualitative improvements can involve the
characteristics of both the product and the production process (for instance the
environmental impact). The need for approval strengthens the integration between
different stages of the product chain.
2.3.5. Public Acceptance
Whether transgenic potato varieties will be grown depends mostly on the attitude
of the public (or consumer). As far as consumer acceptance goes, many issues are
involved. The environmental impact of transgenic potatoes and their safety for human
consumption are the most important. Because there are no definite findings on these
issues, public attitudes remain uncertain.
Public attitudes towards biotechnology have been studied in various countries, as
well as in the European Community as a whole. Most studies showed that transgenic
crops are valued moderately. Of course, the sine qua non is safety for human
consumption and the absence of environmental effects. There seems to be a clear
difference in risk perception for transgenic food products in various EC countries
In an EC study on public attitudes towards biotechnology, it was shown that
most people consider environmental and consumer organizations the most trustworthy
sources of information on the impact of biotechnology. Concerning transgenic potatoes,
the environmental organizations are the most critical. They favor a restrained approach,
because it is still uncertain what the long term environmental impact will be. Consumer
organizations stress the importance of sufficient information for the consumer.
Therefore they favor compulsory labeling.
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To achieve broad acceptance of transgenic agricultural products, a dialogue must
be set up between proponents and critics of genetically modified food products. Only in
this way may consensus be reached on which applications of genetic engineering hold
benefits for all groups in society. Concerning transgenic potatoes, this implies that
biotechnology firms and the potato business on the one hand, and environmental and
consumer organizations on the other hand, must enter a dialogue to discuss which
transgenic potatoes should be developed and introduced.
2.3.6. Transgenic potatoes and the environment
Certain transgenic traits in crop species might alter the plant's ability to invade
natural and semi-natural habitats and cultivated fields. Potato is not invasive of habitats
and the transgenic plant can readily be shown to have similar characteristics before
widespread use. The risk that the gene might be transferred to another plant is a
particular concern in centres of biodiversity of the crop species.
Introgression of DNA from potato to other Solanum species probably occurs
naturally under field conditions. Possibly, hybrids are less locally adapted than the
parents are and fail to persist for many generations. Introgression must also result from
growing potatoes from conventional plant breeding where this introduces new genes to
the agroecological area. This has not been raised as a concern before. One possible way
forward is through the use of male sterility, which occurs naturally in some potato
cultivars. This eliminates the risk of transgene escape via pollen.
2.3.7. Transgenic potato and human health and safety
The safety should be based on the nature of the product rather than on the
method of its production. This is self-evident for potato crops. Conventional plant
breeding inadvertently produced a cultivar that caused gastrointestinal, circulatory,
neurological and dermatological problems associated with alkaloid poisoning. It
reached the marketplace before withdrawal.
Any toxicity or allogenicity associated with a food is universal to all human
populations. Therefore, we consider that those traits in GM food that have gained
approval for use in the developed world are likely to be of value to the developing
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world, providing usage is similar. The nematode resistance we have developed does not
rely on one basis for ensuring food safety; it uses cystatins that are already in the human
diet. Cystatins are ingested every time we swallow because they occur in saliva. They
have also been proposed as food additives and as components of dental care products.
Additionally, the cystatin produced by the transgene will be expressed in roots but not
the potato tuber. The new GM potato would not be approved for entry into the food
chain unless food safety is assured after comprehensive tests.
2.3.7.1. Transgenic resistance to nematodes
The nematode resistance that we have developed involves expression of cysteine
proteinase inhibitors (cystatins) in roots. They act on nematodes by preventing the
cysteine proteinases of the intestine from digesting the protein in the pests’ plant diet.
Cystatins provide resistance to a wide range of nematodes, such as the potato cyst
nematode that fails to thrive and consequently egg production is reduced.
The cystatins involved occur naturally in rice, maize and sunflower seeds, all of
which are in the human diet. Similar proteins occur in potato tubers. It is possible to
construct transgenes with root specific promoters. This ensures that quantitatively
significant levels of the novel cystatin occur in the roots of the transgenic plants and not
in the tuber or the green tissues of the potato.
2.3.8. Effects on non-target organisms
A hierarchical approach has been used to identify the sub-set of non-target
invertebrates that are at risk from the cultivation of nematode-resistant potato. A
histochemical assay identifies those potato associates that have cysteine proteinases
within their gut. It is these species that might be at risk from cystatin expression in
potato plants. Of these, leafhoppers and springtails have been studied in greatest detail.
In addition aphids were examined because previous work has suggested that they are
affected by ingestion of protease inhibitors, although they are thought to lack digestive
proteinases. Those at potential risk are subject to laboratory-based bioassays and
observed under field conditions on transgenic plants. To date, work has concentrated on
the consequences of expressing a cystatin in green tissue. This represents base line
studies involving the worst case scenario.
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The peach–potato aphid was influenced when cystatins were added to its diet but
not when it was fed on plants expressing the same cystatin. This lack of efficacy of the
transgenic plants also occurred with a second homopteran, the leafhopper Eupteryx
aurata. The aphid feeds on phloem and the leafhopper on the contents of mesophyll
cells. The transgenic lines that were effective against a PCN (Globodera pallida) had no
effect on the accumulative numbers of either insect on transgenic potatoes. The
concentrations in the vascular tissue were insufficient to reproduce the adverse effects
on aphids that occurred in artificial diet assays. Clearly, the feeding site and plant
material consumed relative to the pattern of transgene expression help define the risk of
cystatins to herbivorous invertebrates. Root-specific promoters will reduce even further
the risk to above ground insect herbivores irrespective of the plant tissue type they
consume.
There can be no compromise on biosafety, but the environmental consequences of
release must be assessed against the impact of existing agricultural practices. High
standards are being set for transgenic plants. Comparable rigour must be applied to
other components of integrated pest management. For instance there are several
examples of introduced biological agents that pose a threat to native species.
Conventionally bred cultivars can also be unfavourable for natural enemies. Potatoes
expressing a modified form of the -endotoxin of Bacillus thuringiensis. Opposing its
use is inconsistent if a biocontrol agent that consists of the same bacterium expressing a
similar protein is to be allowed. Using the whole microbe rather than one of its genes
presents other risks. Organisms that are indistinguishable from B. thuringiensis colonize
human wounds and a second form, which differs mainly in the toxin gene on one
plasmid, causes anthrax. Consistency demands those who are concerned about
unforeseen risks from GM crops must address the potential hazard of plasmid exchange
between the two microorganisms if one is used as a biocontrol agent.
2.3.9. Starch content and yield increase as a result of altering adenylate pools
in transgenicplants
Starch is the most important carbohydrate used for food and feed purposes and
represents the major resource for our diet. The total yield of starch in rice, corn, wheat,
and potato exceeds 109 tons per year. In addition to its use in a nonprocessed form,
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extracted starch is processed in many different ways. Processed starch is subsequently
used in multiple forms, for example in high-fructose syrup, as a food additive, or for
various technical processes. As a result of its considerable importance, increasing the
starch content of plant tissues has been for many years a major goal, with both classical
plant breeding and biotechnological approaches being taken extensively over the last
few decades.
In most tissues, starch synthesis takes place largely, if not exclusively, in the
plastid. Either glucose-6 phosphate (G-6-P) or glucose-1-phosphate (G-1-P) are
imported into the plastid as, in heterotrophic tissues, is ATP. Subsequently G-1-P and
ATP are converted into ADP-glucose by ADP-glucose pyrophosphorylase (AGPase).
ADP-glucose, the substrate for starch synthases, represents the first committed
precursor for starch synthesis. Subsequent to this reaction, the complex structure of
starch is achieved through the action of a variety of starch-modifying proteins, most
notably branching and debranching enzymes.
Various attempts have been described to increase starch biosynthesis in
heterotrophic storage organs such as corn kernels and potato tubers. Most of these
studies focused on increasing the level of ADP-glucose either by increasing the level of
its immediate precursor in the pathway of starch synthesis or by increasing the activity
of the ADP-glucose pyrophosphorylase by modifying its allosteric properties. Although
the former approach failed, the expression of an ADP-glucose pyrophosphorylase
displaying modified allosteric properties led to substantial increases in starch content.
More recent studies have shown that the adenylate supply to the plastid is of
fundamental importance to starch biosynthesis in storage organs such as potato tubers.
Overexpression of the amyloplastidial ATP:ADP translocator resulted in an increased
starch accumulation, whereas antisense inhibition of the same protein resulted in a
reduced starch yield. Furthermore, incubation of tuber disks in adenine resulted in a
considerable increase in cellular adenylate pool sizes and a consequent increase in the
rate of starch synthesis. The enzyme adenylate kinase (EC 2.7.4.3) catalyzes the
interconversion of ATP and AMP into ADP. Because adenylate kinase is described as a
crucial enzyme maintaining the pool sizes of various adenylates at equilibrium, it
represents an interesting target for modulating the adenylate pools.
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It is shown that downregulating the activity of the plastidial isoform of adenylate
kinase has substantial effects on the pool size of the various adenylates and, most
importantly, leads to an increase in total tuber yield above that found in wild type plants
(of 39%) and an increased starch content per gram fresh weight (by 60%) above that
found in wild type plants. These data thus illustrate that the modulation of the adenylate
kinase activity represents a useful strategy for increasing formation of one of the most
important resources for both human and animal diet.
2.3.9.1. Cloning of a cDNA encoding plastidial adenylate kinase from potato
Screening a Solanum tuberosum cDNA library using a maize cDNA encoding
adenylate kinase, a clone of 888 base pairs was isolated (subsequently named StpADK;
GenBank accession no. AF411937). Sequence analysis of the potato adenylate kinase
revealed an open reading frame of 284 amino acids. Comparison with the functionally
characterized adenylate kinases of maize and rice revealed 97% and 74% identity with
the plastidial isoforms and a much lower homology with both the cytosolic and/or
mitochondrial isoforms (Fig. 2A).
StpADK bears an N-terminal domain that contains a high degree of
hydroxylated amino acids (predominantly serine and threonine residues) characteristic
of a plastidial transit peptide, indicating a plastidial location for the enzyme encoded by
the StpADK cDNA.
Analysis of mRNA northern blots using the StpADK cDNA as a probe indicates
constitutive expression of the gene, with the transcript present at approximately
equivalent levels in young and mature leaves, stems, roots, stolons, and developing and
mature tubers.
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Fig. 2A
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2.3.9.2. Transgenic plants display a wild-type growth and developmental
phenotype
The full-length cDNA encoding plastidial adenylate kinase was cloned in the
antisense orientation into the transformation vector pBinAR-Kan between the
cauliflower mosaic virus (CaMV) 35S promoter and the ocs terminator (Fig. 2B). Then
it is transferred 60 transgenic potato plants obtained by Agrobacterium tumefaciens–
mediated transformation to the greenhouse.
Screening of these lines for a reduction of the StpADK-encoded mRNA yielded
six lines that displayed a substantial reduction on the transcript level in both leaf (Fig.
2C) and tuber extracts. Using an assay optimized for potato tissue (Table 2), it is
observed that the total adenylate kinase activity was reduced 67% relative to that found
in wild type in leaves and to a similar extent in tubers. Furthermore, analysis of
chloroplasts isolated from leaves of both wild-type and transgenic plants for adenylate
kinase showed that this loss in activity was localized to the plastid—a result in
agreement with the deduced subcellular localization based on sequence (Fig. 2A). There
was little change in the activities of other enzymes of the starch biosynthetic pathway,
with only the AGPase activity of line ADK-24 being significantly different (higher)
from that found in the wild type. When the transgenic plants grew in the greenhouse
side by side with wild-type controls, there were no observable phenotypic changes
concerning either the aerial part of the plant or the tuber size, number, or morphology in
lines ADK-20 or ADK-4; however, there were significant increases in the total tuber
yield in lines ADK-2 and in both tuber number and yield in line ADK-24 (Table 3).
Furthermore, the specific density of the tubers was elevated in lines ADK-20, ADK-2,
and ADK-24, indicating that these lines are most probably characterized by a higher
starch content.
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Fig. 2B
16
Fig. 2C
17
Table 2
Table 3
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2.3.9.3. Reduction of StpADK activity results in a general increase in the
adenylate pools
Adenylate kinase catalyzes the interconversion of ATP, ADP, and AMP. To
analyze the effects of a reduced adenylate kinase activity on adenylate pools, it is
determined the steady-state levels of all three metabolites directly involved in the
reaction and of ADP-glucose. The reduction in the activity of the plastidial adenylate
kinase led to clear changes in the levels of the various adenylate pools (Fig. 3). Only
one of the most strongly inhibited lines (ADK-2) exhibited a significant increase in
ATP; however, the levels of both ADP and AMP increased significantly in all lines.
Overall, though, the largest changes were observed in the ADP-glucose content, which
increased between three- and tenfold in the transformants but did not correlate closely
with the change in adenylate kinase activity. However, this is not surprising given that
ADP-glucose represents neither a direct substrate nor a product of the reaction catalyzed
by adenylate kinase. When taken together, these data demonstrate that both the total
adenine nucleotides and the total adenylate pool sizes were increased in the transgenic
lines. These changes were accompanied by a decreased ratio of ATP to ADP but not by
changes in the adenylate energy charge18 or in the concentrations of glycolytic
intermediates, uridine nucleotides, or phosphate; moreover, they were representative of
values obtained over separate harvests.
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Fig.3
20
Fig. 4
21
Fig. 5
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2.3.9.4. Inhibition of adenylate kinase results in an increased tuber starch
content
ADP-glucose is the direct precursor for starch biosynthesis. Given the increase
in ADP-glucose described earlier, it is decided to analyze the starch content in the
various transgenic lines to determine whether or not this increased ADP-glucose
concentration results in higher starch levels. Figure 4 illustrates clearly that all plant
lines analyzed had an increase in starch content that was negatively correlated with the
residual adenylate kinase activity. This increase is important in the most strongly
affected lines (ADK-2, ADK-4, and ADK-24), the starch accumulation in line ADK-24
being >60% above that found in the wild-type control. Similar results were obtained
from an additional two harvests from greenhouse-grown plants. When taken together,
these data demonstrate a link between the reduction in adenylate kinase activity and
changes in the adenylate pools on the one hand, and the increase in starch content on the
other.
2.3.9.5. Performance of the adenylate kinase antisense plants under field
conditions
In an additional experiment, the adenylate kinase antisense plants grew from
size-normalized seed tubers in the field alongside untransformed control plants and
followed their growth. Although there were no major changes either in the aerial portion
of the plants or in the earliness of the crop, differences were found in the tubers (Table
3). Most importantly, total tuber yield was significantly increased to between 65% and
85% above that found in wild type in all three of the lines tested.
Interestingly, this does not correlate with an increased tuber number; note that
line ADK-24, which exhibits the largest increase in yield, displayed the same tuber
number as wild type under the conditions of this study. Because the increase in yield
was also accompanied by significant increases in tuber density, estimates of the starch
content per plant were found to be double that found in the wild-type lines. Whereas
these trends were also apparent (though considerably weaker) when the transgenics
were compared to a second batch of wild-type plants grown in a different plot in the
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same field, it is important to note that large differences between the two field plots of
wild type were also observed.
2.3.9.6. Inhibition of adenylate kinase results in increased amino acid levels
in tubers
ATP levels could be expected to have a direct bearing not only on starch but also
on amino acid levels. Because it has been recently demonstrated that the tuber is
capable of de novo biosynthesis of all amino acids. Analysis of the pool sizes of the
various amino acids revealed significant increases in several amino acids, notably up to
twofold changes in alanine, histidine, isoleucine, methionine, phenylalanine, and
tyrosine and up to fourfold changes in leucine and tryptophan (Table 4). These changes
were noticeably more prominent in the transgenic lines exhibiting the strongest
reduction in the plastidial adenylate kinase enzyme activity, in particular in lines ADK2 and ADK-24. As expected on the basis of the increases in pool size of individual
amino acids, the total amino acid content of the tubers also exhibited a trendwise
increase, although because of the larger variation this increase was not statistically
significant considerable success, many of the push approaches have failed despite
successful changes in some or all of the required metabolic precursors. A general
characteristic of these approaches has been that the main emphasis was on trying to
increase the concentration of metabolites that are specifically involved in a given
biosynthetic pathway. Surprisingly, metabolites involved in multiple biosynthetic
reactions have received little attention to date. The adenylate pools fulfill this criterion,
and because adenylate kinase equilibrates the concentrations of AMP, ADP, and ATP,
the activity of the plastidial isoform of this enzyme is modulated. As described under
increase in starch can essentially be explained as a direct consequence of the increase in
ADP-glucose. Furthermore, the increases in the concentrations of several of the amino
acids could also be explained by the changes in the adenylate pools. The plastid is also
the predominant location for amino acid biosynthesis, and ATP is involved in the
synthesis of many amino acids—most notably arginine, methionine, histidine, and
tryptophan, but also lysine, isoleucine, phenylalanine, and tyrosine. These are exactly
the amino acids that show the most profound increases in the transgenic lines analyzed.
It is still unclear by what exact mechanism the reduction of plastidial adenylate
kinase brings about such important changes in the above-mentioned parameters.
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Moreover, given that the constitutive CaMV 35S promoter was used to drive the
expression of the transgene, it cannot be excluded the possibility that changes in other
parts of the plant contribute to the biochemical effects seen in the tuber. Furthermore,
the interpretation of changes in adenylate levels is incredibly complicated because of
the multiplicity of reactions in which they are involved, and it is therefore not possible
to determine whether the reported changes are direct or indirect consequences of the
reduced adenylate kinase activity. Despite these limitations, it is clear that the reduction
of this enzyme activity in the potato yields important results when considered from a
biotechnological standpoint.
Table 4
Adenylate kinase is ubiquitous. It has been demonstrated to play a crucial role in
both yeast and mammalian systems, knockout mutants of which display strong
phenotypes characterized by disease and lethality. However, the predominant direction
of the reactions catalyzed by adenylate kinase reactions is unknown. Kinetic
characterization of the plant enzymes purified to date reveals that adenylate kinase
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displays high affinities, in vitro, for the substrates of both the forward (ATP-generating)
and reverse (ATP-consuming) reactions, and as such is consistent with suggestions of
the free reversibility of this enzyme. Under in vivo conditions, at least in the potato
tuber amyloplast, adenylate kinase acts in the ATP-consuming direction, competing for
ATP both with ADP-glucose pyrophosphorylase and with plastidial pathways of amino
acid biosynthesis.
As result, antisense repression of the plastidial adenylate kinase led to increases
in starch to 60% above wild type, in tuber yield to 84% above wild type, and in certain
amino acids of between two- and fourfold. The increase in starch content represents the
largest increase yet observed in a transgenic approach (being in excess of the increase in
starch per gram fresh weight, of 35% above wild type.), particularly when one considers
that this occurs without the usually expected yield penalty but rather in combination
with a increase in harvestable biomass.
It is tempting to speculate that these increases are caused directly by the
increases in the adenylate pool. Starch biosynthesis takes place in the amyloplast, and
various data have shown that ADP-glucose is the only committed precursor for starch
biosynthesis. It therefore follows that the transgenic potato plants led to observable
increases in adenylate pools, in starch content, and in the concentration of some amino
acids, as well as a slight increase in total tuber yield.
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Potato root with various stages of the potato cyst nematode life cycle. These can be
seen at various stages of tanning, from immature white cysts to mature ‘tanned’ cysts.
A cyst of Globodera spp. broken open to reveal eggs and infective juveniles. Each
potato cyst nematode can contain 200–400 eggs, and can persist for long periods of time
imposing large crop losses.
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