Barley and wheat

Phosphate metabolism
Amino acid metabolism
Iron and zink metabolism
Endosperm cell walls
Risk assessment
Improved transformation techniques

Phosphate metabolism

Generation of Wheat with Increased Phosphate Bioavailability

Phosphorus is an essential nutrient for living cells. The metabolic form of phosphorus is mainly phosphate. In plants, phosphate is absorbed from soil fluids by the roots by means of specific phosphate transporters in the cell membrane. When absorbed, the phosphate can enter into the cell’s metabolism or be stored in the vacuole in the form of phytic acid. During grain filling, up to 80% of the total amount of phosphate is incorporated as phytic acid, which in practice exists as a mixed salt (phytin) composed of i.e. iron and zinc. In natural ecosystems phosphorus will return to the soil when the plant withers and the plant material is decomposed by biological and chemical processes in the soil. As phytic phosphorus is practically indigestible to monogastric animals, the phosphorous cycle is further affected by contributions from livestock production in cultivated ecosystems. This results in a multiplication of the amount of phosphate returned to the soil and in consequence an increased pollution of the aquatic environment.

Amino acid metabolism

Today cereals provide a very significant proportion of both human and animal diets despite the fact that most grain are to a greater or lesser extent deficient in a number of essential nutrients. A primary problem, is the low level of essential amino acids such as lysine, methionine and threonine in the major cereal storage proteins. Barley is no exception and because of its central position in Danish agriculture it is of particular interest. In animal feed compositions based on barley grain, industrially derived lysine and threonine are added to obtain a balanced nutritional diet. However, the essential amino acids must be added at additional cost. Other non-essential amino acids such as glutamine and proline are present in excess in the major storage proteins and create a different problem. These amino acids, when digested by the animal, release non-utilisable nitrogen. This nitrogen is excreted in the urine, creating a significant environmental load, especially on and around pig farms.

We aim to modify amino acid metabolism in the developing barley seeds by genetic manipulation in order to improve the nutritional value of the grain. To channel the amino acid flow towards the desired direction, we are trying to increase the asparagine synthetase activity in different parts of the plant and asparaginase activity in the seed. Our approach to address the nitrogen contamination problem is to interfere with storage protein synthesis and the free proline availability in barley seeds.

The practical aim is to create barley seed that is nutritionally more beneficial for animals. In the process we are also collecting new knowledge about amino acid metabolism and its regulation during seed development.

Metabolism of iron and zink

The group is working with improvement of the bioavailability of iron, and zinc in grains

Depositing and Transport of Iron and Zinc in Wheat

In the developing countries many poor families live on a simple diet primarily consisting of basic food (such as rice, wheat, and maize). While these foods are good sources of in particular carbohydrates, they have a low content of iron and zinc.
It is said that 2 billion people are in danger of developing iron deficiency at the moment. Babies, children, and women of childbearing age are particularly vulnerable. The primary symptom is anaemia, which is caused by a too poor formation of haemoglobin owing to an insufficient supply of iron. In plants, iron is an important constituent of various redox and iron-sulphur enzymes and at the same time it is important for the formation of chlorophyll (leaf green). Zinc deficiency has several consequences, such as complications during pregnancy and at birth, low birth weight, and slower growth in combination with diarrhoea, reduced immune response, and increased sickliness. More than 300 enzymes require zinc as an auxiliary factor. Zinc deficiency can be just as widespread as iron deficiency, but it is much more difficult to diagnose.


Arabinoxylan is the main component of cell walls in wheat endosperm. In collaboration with The Royal Veterinary and Agricultural University and Odense University we plan to make a detailed characterisation of the structure and degradation of arabinoxylan, and evaluate the importance for baking quality. Our contribution to the project includes transformation of wheat with genes encoding various hydrolytic enzymes, either expressed throughout the plant or specifically in the endosperm. Our collaborators will then characterise the material using biochemical and mass spectrometrical methods.

Risk assessment - molecular studies on unintended side-effects in transgenic plants

The safety of transgenic plant cultivars in relation to human and animal consumption and in relation to the spread of genes into the natural environment is of big public concern. One part of the concerns addresses the uncertainty about unattended phenotypic side-effects caused by the transformation process. The argument is that the introduction of a transgene into a plant might have effects on the expression of other genes, either directly - due to perturbations by the random insertion process, or indirectly - due to cross-talking between different metabolic pathways of the plant in which the encoded product of the transgene may work. In order to address the issues raised by these concerns, there is a great need for the development of techniques which allow for a more global assessment of plant compound profiles in transgenic plants. Such techniques would be extremely valuable both as scientific tools during the development of transgenic plant cultivars as well as for the official evaluation of these materials. In this context we have focussed our work in this area on the development of cDNA microarrays to be used in studies of the global gene expression profiles, in the first place of transgenic wheat plants developed at Research Centre Flakkebjerg.

Improved transformation techniques - Microarrays

Gene expression analysis is performed using cDNA microarrays fabricated in-house:

Probe generation and spotting of microarrays

PCR products are produced from available cDNA clones, using vector primers flanking the cDNA inserts. PCR products are quality checked on agarose gels before spotting, and appropriate amounts are spotted onto microarray slides using a QArrayMini ( www.genetix.co.uk ) microarray spotter.

Target preparation and hybridization

PolyA+ RNA is isolated from frozen and ground plant material using paramagnetic Dynabeads ( www.dynal.no ). cDNA target populations are reverse transcribed from the isolated mRNA, and they are labelled using the indirect labelling method where reactive esters of Cy3 and Cy5 dyes are coupled to aminoallyl-dUTP. Hybrizations to the spotted microarray slides are performed under LifterSlips ( www.eriemicroarray.com ) in closed hybridization chambers.

Scanning and signal quantification

Scanning of hybridized slides is performed on a ArrayWoRx BioChipReader (Applied Precision), which is a CCG camera based system. Quantification of spot intensities is done using the quantification software, softWoRx, coming with the scanner.

Data storage and data analysis

For storage and handling of the microarray data an instance of BASE (BioArraySoftwareEnvironment – http://base.thep.lu.se/ ) is installed on a server in the laboratory. The rawdata from the scanning can be uploaded to BASE which provides a storage platform where the data is readily accessible and where it can be organized and processed in a comprehensive way. For stastistical analysis of experiments which might include several microarray hybridizations, analysis software from the Bioconductor package ( www.bioconductor.org ) is applied, especially LIMMA which include a range of facilities to preprocess and analyse microarray data.

cDNA microarrays from the following species are currently under preparation:


9K wheat cDNA microarray.

A 9K wheat unigene set was kindly made available by Keith Edwards, Bristol University ( www.cerealsdb.uk.net/ ). These clones originate in more than 40 different cDNA libraries, mostly from the developing seed of wheat. PCR products of the cDNA inserts were generated using flanking primers. Following check for quality and integrity on agarose gels, the PCR products were spotted in two replicates onto aminosilane coated slides (Quantifoil, QMT slides). Thus, the slides contain approx. 20000 spots.



Last updated: Wednesday 08 October 2008