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FAIR-CT95-0568
Production of novel starch polymers in maize, wheat, barley and potato |
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Proposal No: | FAIR-CT95-0568 |
| Date Prepared: | July 2001, April 1998, February 1997 | |
| Source: | Final Report
Abstract and
Executive Summary Second Annual Progress Report First Annual Progress Report |
Final Report Abstract
Source: Final report of 31 March 1999
Consortium: This project was coordinated by Centre National de la Recherche Scientifique, Relations Structure-Fonction des Constituants membranaires (France) together with Institut National de la Recherche Agronomique, Centre de Recherches de Nantes, Biochimie et Technologie des Glucides (France), Ulice SA (France), Danisco A/S, Maribo Seed, Biotechnology (Denmark) and Cooperative verkoop- en Produktievereniging van Aarappelmeel en Derivaten, Avebe Research & Development, Department of Biotechnology (The Netherlands).
Abstract
Objectives: The ultimate aim of this project was the generation of new starch polymers in the most important EU crops. This could only be achieved if the understanding of structural specification in starch granules was improved and a full understanding of how technologically useful substructures are produced obtained. The project thus aimed both to generate such knowledge and using this to produce new polymers in potato, maize, barley and wheat.
Activities: The work was divided into the following four majors tasks:
Three breeding programmes involving wheat, maize and potato were integrated into these tasks with the following aims.
Results: The breeding programmes were largely successful. Low amylose wheat was indeed produced and confirmed in field trials. In maize, double mutant combinations involving the waxy, amylose extended and sugary2 loci were selected and subjected to intensive structural characterisation. In potato, monoploid lines were successfully created permitting selection of mutants in a haploid genetic background. In addition over-expression of the barley starchy endosperm ADP-glucose pyrophosphorylase in potato tubers led to significant increases in starch dry matter content. All these breeding programmes have long-term objectives and are being continued, although the project funding has now ceased.
Several approaches, dependent on basic science, were also adopted in order to improve understanding of starch metabolism. These included:
Single celled organisms, such as the bacterium Escherichia coli and the monocellular green alga Chlamydomonas reinhardtii were used as model systems to develop a greater understanding of the structural specification of amylose and amylopectin. In E. coli single, double and triple expression systems were constructed with combinations of cDNAs corresponding to the major enzymes of starch biosynthesis cloned from potato tubers. All these bacteria produced glycogen-like polymers suggesting that the additional determinants required for starch biosynthesis had to be obtained from plants. While Chlamydomonas proved to be a poor system for the expression of foreign genes it became a very successful model system when used to seek these additional unknown determinants.
In particular, as a result of this project, debranching enzymes were defined as mandatory elements of the starch biosynthetic pathway. In addition Chlamydomonas starch granules were shown to contain a very high specific activity granule-bound starch synthase. These starch granules could thus be used in an in vitro system to investigate the mechanism of amylose synthesis within the granule. It was found that granule-bound starch synthase could extend a chain of a pre-existing amylopectin molecule. Synthesis was terminated by hydrolytic cleavage and the mature amylose released within the granule.
Since the availability of the substrate of starch biosynthesis was suspected to have different effects on the contributions of the various starch synthases to the mature granule structure, the level of ADP-glucose was artificially lowered. In pea or Chlamydomonas, this was done by introducing mutations for one of the genes encoding the enzyme responsible for ADP-glucose synthesis. In potato this was done by lowering the level of the corresponding mRNA. It was found that amylose synthesis responded swiftly to such modifications and that increasing or decreasing ADP-glucose led to selective increases or decreases in the rates of amylose biosynthesis. The high phosphate content of potato tuber amylopectin is largely responsible for making this source of starch particularly attractive for industrial uses. One of the aims of this project was to increase understanding of the mechanism of potato starch phosphorylation, which is a prerequisite to introducing this desirable trait into other major crops. Potato starch was found not to be phosphorylated through a phosphorylated sugar nucleotide intermediate but rather through the action of a previously unknown protein, which is selectively bound to potato starch.
A lot of effort was put into dissecting, at the molecular level, the structural/functional relationships of granule-bound starch synthase, soluble starch synthase and branching enzyme. Expression of full size enzymes was achieved successfully in E coli. Mutations were introduced in both starch synthases and branching enzymes. Artificial starch synthase genes were built by mixing various combinations of C-terminal and N-terminal domains. This strategy was not only informative as far as the function of the normal enzymes were concerned. In addition novel hybrid starch synthase genes were also successfully reintroduced and expressed in potato tubers. The transformed potatoes produced starches with altered structure proving that such novel strategies can lead to the production of novel starch polymers.
Results of the program were disseminated at an international conference that was organised by the co-ordinator during September 1999 near Marseilles (France). This meeting was attended by around 100 scientists from all over the world, including all the major European and non European companies active in plant breeding research.
Objectives
The ultimate goal of this program is the generation
of new starch polymers in the major EU crops. This can only be achieved if we
improve our understanding of structural specification in starch granules and if
we get a full understanding on how technologically useful substructures are
produced. This project aims both at generating such knowledge and at producing
new polymers in potato, maize, barley and wheat
Description of work
The objectives of the program will be
reached by carrying out the following tasks:
Task 1:
To improve our understanding of structural
specification in starch granules, with the following sub-tasks:
Task 2:
To establish the correlation between important
technological attributes of starches and defined polysaccharide substructures,
with the following sub-tasks:
Task 3:
To get a full understanding on how
technologically useful substructures are produced, , with the following
sub-tasks:
Task 4:
To devise means by which novel structures can
be produced and to achieve production of new starch polymers, with the
following sub-tasks:
Progress
The progress made, to date, is summarised on a task by
task basis below.
Sub-task 1.1:
During the first year of the program
plants containing transposable Ds elements inserted in the vicinity of genes
known to be involved in starch biosynthesis were successfully selected. During
the second year 17 genotypes were selected and the offspring of crosses with
wild-type potatoes was successfully analysed. This offspring is now being used
in a second round of crosses aiming at introducing the transposase gene to
activate the mutagenesis. The same offspring is also being used to introduce the
transposase gene directly by transformation. To maximise the chances that
cultivars of interest will indeed be produced, a random mutagenesis approach
involving monoploid selection is simultaneously being undertaken
Sub-task 1.2:
Introduction of potato cDNAs encoding
various starch synthases have been successfully introduced in the Chlamydomonas
reinhardtii plastid genome during the first year of the program. The
constructs and the plastidial transformants have been thoroughly checked and
characterised during this reporting period. In all cases despite the presence of
functional algal plastid promoters no expression of the potato starch synthases
could be detected. The focus of the work was then turned to the collaborative
cloning and characterisation of the endogeneous Chlamydomonas starch
synthases. Successful cloning of 3 distinct Chlamydomonas starch
synthases was achieved. During the first reporting period, 2 barley cell lines
were successfully established and monitored for starch synthesis. During this
second reporting period detailed structural analysis were performed on these
cell lines and yielded one highly crystalline and one amorphous polysaccharide
accumulating cell line. Full length cDNA cloning of the barley branching enzymes
which is a prerequisite for their planned introduction in the two aforementioned
cell lines has now been successfully achieved Their expression pattern in the
barley endosperm was monitored. Expression studies were also performed for
starch synthases in the recipient barley cell cultures. The absence of
expression of SSS55 (a specific barley soluble starch synthase) in the cell
cultures precludes their use for the originally planned antisense RNA inhibition
experiments. Recent methods authorising the introduction of antisense constructs
into whole plants have appeared during this reporting period. Since these
methods appear to be as fast as the planned introduction of genes into the
barley cell cultures, the constructs planned for expression will be directly
introduced into whole plants. If successful this could lead directly to the
production of useful and novel barley starches.
Sub-task 1.3:
During the first reporting period
construction of plasmids allowing expression of single potato starch
biosynthetic enzymes into E. coli cells defective for glycogen
biosynthesis was achieved. During this second reporting period constructs were
built allowing the simultaneous expression of several such genes. Glycogen from
the recombinant E. coli strains containing these constructs was extracted and is
currently under analysis. In addition genes encoding the barley and
cyanobacterial branching enzymes and the barley SSS55 were introduced and
successfully expressed. Polyglucans are now being extracted from the bacterial
recombinant strains.
Sub-task 2.1:
Starch from the single and double mutant
lines (4th backcross generation) that were produced during the first reporting
period were used for detailed structural analyses. The structures proved to be
original in that they displayed a crystalline organisation (V-amylose) that is
yet to be reported within native starch granules. An additional generation of 6
of the most promising distinct double mutant genotypes has been produced in 3
distinct sites. The starch has been extracted and passed on for detailed
structural analysis.
Sub-task 2.2:
Because of the low recovery frequency of
wheat transformants that was obtained at the end of the first reporting period,
efforts have concentrated on the production of antisense plants with
down-regulation of the amylose biosynthetic enzyme GBSSI. Of 26 distinct
transgenic lines one particular line displayed reduced amylose content during a
first round of analysis and will have to be confirmed.
Sub-task 2.3:
Task 2.3 was successfully completed
during the first reporting period. Results have been disseminated through
publication during this second reporting period.
Sub-task 2.4:
The analysis of the starch from low
ADP-glucose containing plants and algae is proceeding. The reduction of amylose
synthesis witnessed in mutants from Chlamydomonas with reduced ADP-glucose
pyrophosphorylase and plastidial phosphoglucomutase was confirmed. The X-ray
diffraction pattern of these starches at variance with our expectations
displayed surprisingly good crystallinity of the A-type. Antisense potato plants
with reduced ADP-glucose pyrophosphorylase activity were produced. In contrast
to peas and Chlamydomonas these plants did not display the expected reduction in
amylose content. The starch from the antisense plants has now been passed on for
detailed structural analysis.
Sub-task 2.5:
During the first reporting period
evidence was provided for the cytosolic localisation of the barley endosperm
ADP-glucose pyrophosphorylase and constructs were made allowing expression of
the two enzyme subunits in insect cells. During this second reporting period
transgenic potato lines expressing the high activity barley enzyme were produced
and are currently being analysed. AGPase production in insect cells was
successful. As expected only the small subunit homotramer was active while the
heterotetramer displayed sensitivity to orthophosphate inhibition and high
catalytic activity in the absence of 3-PGA.
Sub-task 3.1:
During the first reporting period a
cassava cDNA encoding GBSSI was successfully introduced in an amylose-free
potato line. During this reporting period, the transformed plants were
characterised in detail. The substitution of the native GBSSI by the cassava
enzyme led to modification not only in amylose content but also in structures of
both amylose and surprisingly also amylopectin. The technological attributes of
the most productive amylose synthesising transgenic plant were intermediate
between those characterising amylose-free and wild-type potatoes.
Sub-task 3.2:
During the first reporting period,
phytoglycogen producing mutants were used to study the determinants of amylose
synthesis in vivo. This led to propose a model explaining the basic features of
amylopectin synthesis in plants. During this second reporting period an
additional collaborative effort between participants 1 and 4 has led to the
establishment of an in vitro amylose synthesis system through GBSSI. Particular
mutant backgrounds were used to maximise GBSSI specific activity within the
granule and to minimise amylose content prior to in vitro synthesis. This system
is expected to lead to additional insights into the physiologically active
determinants of amylose synthesis in plants.
Sub-task 3.3:
Significant progress has been made as to
the localisation of the phosphorylated residues in amylopectin. The presence of
high levels of phosphorylation in Curcuma starch was associated with an increase
of the mean DP to 19. Techniques were set-up to selectively study the
chain-length distributions of phosphorylated glucans. On the other hand
introduction of the E. coli ADP-glucose pyrophosphorylase in substitution for
the native plant enzyme was achieved in potato. The plants did not display any
significant reduction in phosphate content ruling thus out that potato
ADP-glucose pyrophosphorylase would distinguish itself by the ability to produce
ADP-glucose-6phosphate in vivo.
Sub-task 4.1:
During the first reporting period hybrid
bacterial glycogen and plant granule-bound starch synthases have been
constructed together with hybrid enzymes involving distinct domains of the
granule-bound and soluble potato starch synthases. In this second reporting
period considerable progress in our understanding of the molecular function of
the starch synthases has been achieved. The processivity of GBSSI with respect
to amylose synthesis was located on the N-terminal part of the enzyme while
activation by amylopectin was demonstrated to depend on the C-terminal portion.
In addition recombinant GBSSI which was known to display low activity as a
soluble enzyme was shown to be substantially activated by very high
concentrations of amylopectin in vitro.
Sub-task 4. 2:
During the first reporting period potato
and E. coli recombinant branching enzymes were over-expressed in E. coli. During
this second reporting period enzyme purification methods had to be developed to
maximise the yield in recombinant enzyme. In addition site-directed mutagenesis
including point mutations and deletions have been initiated on these constructs.
In barley, protocols achieving satisfactory transgenesis were set -up as a
preliminary to the introduction of branching enzyme genes from various sources
No work was planned under tasks 4.3 and 4.4 for the second reporting period.
Achievements and future actions
During this reporting period
particular progress has been made in our understanding of amylose synthesis in
plants. Both the successful expression and molecular characterisation of
recombinant potato GBSSI and the setting-up of in vitro amylose synthesis are
expected to yield major insights into the understanding of amylose biogenesis by
the next reporting period. In addition significant progress has been made in
understanding the origin of starch phosphorylation as well as locating more
precisely the position of the phosphate groups. Breeding programs aiming at the
production of novel maize, wheat and potato starches are proceeding normally.
Novel starches displaying V-type diffraction patterns as well as waxy wheat
obtained through antisense technology are announced in this report and will have
to be confirmed in the next reporting period.
INTRODUCTION
This project aims to develop novel starch polymers, using a multidisciplinary approach integrating
aspects of both applied and basic science, in the major EU crops (maize, wheat, potato and barley).
The work has been divided into a number of Tasks. The first of these aims to improve understanding
of structural specification in starch granules. Activities include the production of novel mutant structures
by transposon tagging of diploid potatoes, the setting up in Chlamydomonas and barley
of starch synthesising systems in order to produce novel structures by heterologous gene expression and
setting up in E.coli of starch synthesis reconstitution systems. The second task aims to
establish correlations between important technological attributes of starches and defined polysaccharide
substructures. The work includes establishment of correlations between starch structures and
technological behaviour in maize and wheat cultivars selected by the industrial participants, as well
as those between chain length distribution of amylopectin and crystal packing. In addition the
structure, composition and technological behaviour of low ADP glucose containing plants will be
studied and attempts will be made to increase the starch dry matter content using barley starchy
endosperm ADP glucose pyrophosphorylase subunit genes. The third task is to establish a full
understanding on how technologically useful substructures are produced. This will include studies
of amylose production in potato aimed at building novel polymers using transgenic plants containing
various GBSS gene constructs, an understanding of the origin of the amylose fraction and the
pathway of starch phosphorylation in potato. Task four aims to devise means by which novel
structures can be produced and to achieve production of new starch polymers through genetic
engineering of novel hybrid starch synthases or in vitro mutagenesis of the synthases and branching
enzymes followed by expression in higher plants as well as heterologous expression and overexpression
of cyanobacterial or barley branching enzymes in potatoes and cereals. Finally, the novel starch
polymers will be characterised in terms of structure and technical parameters.
ACHIEVEMENTS
The work anticipated for the first year of the project was, in general, completed successfully.
Thus the generation of transgenic potatoes containing Ds elements in the neighbourhood of several
genes encoding enzymes involved in starch biosynthesis was achieved in diploid potatoes. Mobilisation
of the transposons by trans supplied transposase is presently being undertaken by crossing and
transformation. The constructs required to express potato cDNAs (corresponding to several starch
synthases) in Chlamydomonas reinhardtii plastid have been made and tested in
E. coli. Surprisingly GBSSI, the amylose biosynthetic enzyme, which previously failed to
express its activity in E.coli was successfully expressed. The Chlamydomonas
reinhardtii recipient strains defective for GBSSI, GBSSI and SSSII and SSSII only were
constructed by crossing. The potato constructs were successfully introduced in those
Chlamydomonas reinhardtii strains by particle gUD rnediated transformation.
The recipient barley starch synthesising system has been established. Introduction of cyanobacterial
glycogen branching enzyme in this system awaits the building of the suitable construct. Finally all
constructs that were programmed in E. coli cells synthesising high levels of ADP
glucose but otherwise defective for the expression of all other genes involved in glycogen biosynthesis
were built and successfully expressed. These involve potato phosphorylase, starch branching enzyme
I (SBEI), and 3 forms of starch synthases (GBSSI, SSSIII, SSSI)
The maize breeding program (4th backcross generation) involving selection of single or double mutant genotypes was followed through. A NIR (near infrared) test was developed to select lines from a single grain allowing rapid identification of the desired single or double mutant genotypes. The maize mutant genes under selection consist of du (dull), ae (amylose extender) and wx (waxy). Samples of the lines of interest are now becoming available for structural analyses. The wheat breeding program has required development of transformation procedures. Constucts aimed at down regulating GBSSI and SSS have been built. Constructs aimed at overexpressing the E. coli glycogen synthase and branching enzyme and both of the pea branching enzymes have also been built. Wheat transformation experiments involving these transgenes have been performed. Transgenic plants are currently being detected through PCR analysis. l5g of seed has been collected from the first transgenic plants. X ray diffraction has been applied to various Chlamydomonas reinhardtii strains harbouring different combinations of granule bound and soluble starch synthases. The results unambiguously identifies Chlamydomonas SSSII as the major enzyme involved in building the amylopectin crystal. Hybrid potato granule bound starch synthase and E. coli glycogen synthase are being constructed. Five chimerical constructs will be available soon for expression, first in yeast and then in potato. A number of chimerical proteins involving parts of starch synthase domains from potato GBSSI and SSSII have been successfully expressed in E. coli. Most chimerical proteins exhibit very little activity. However and most importantly the low GBSSI activity seen in E. coli seems to be stimulated by oligosaccharides. Chemically modified maltose has been synthesised to investigate this effect further. As a first step towards assaying mutant or hybrid branching enzymes in E. coli, prior to their use in plants, suitable bacterial recipient strains were obtained and both potato branching enzyme and E. coliglycogen branching enzyme were overexpressed. Both enzymes will be purified and used for crystallisation. The chemical synthesis of linear and branched oligosaccharides to be used for investigations dealing with the substrate specificities of branching enzymes and starch synthases is proceeding as expected.
CONCLUSIONS
In general the project has proceeded as expected during the first year. However, a number of
unexpected and important results have been obtained. These include the high level of expression
of GBSSI in E. coli and the stimulation of this enzymes by oligosaccharides, the
localisation of AGPase (ADP glucose pyrophosphorylase) in the cytosol of cereals and the finding
of a glucan trimming mechanism required for starch synthesis in plants.
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