Some plants such as legumes can obtain their nitrogen from the atmosphere via symbiotic nitrogen fixation, while other plants, including most crops, depend on the availability of nitrogen resources in the soil. Farmers usually try to increase crop yield by applying nitrogen-containing fertilizers to the soil. However this method is not only costly, but also harmful to the environment. Therefore, boosting the efficiency of nitrogen assimilation and utilization in higher plants will have significant economic and environmental benefits.

Prof. Lam Hon-ming of the Department of Biology has since 1998 received a total of over HK$1,030,000 from the Research Grants Council to study an important nitrogen metabolic enzyme, namely, asparagine synthetase (AS). This enzyme catalyzes asparagine, one major function of which is to transport and store nitrogen according to the plant's need. It can also re-allocate nitrogen during specific developmental stages and environmental changes. For example, during seed development, it helps transport nitrogen to the seeds, and under stress conditions, it stores nitrogen so that it is not wasted. In short, asparagine has a very close relationship with plant growth and development.

There are, however, major obstacles in biochemical and physiological studies for the thorough understanding of the role of asparagine metabolism. This is mainly because the purification of plant AS enzymes is difficult. Besides, the production of AS in higher plants is governed by a small gene family (three genes: ASN1, ASN2, and ASN3 encoding for three isoenzymes) instead of one gene. Although their ultimate role is to produce asparagine synthetase, the protein structures they encode and their bioactivities may be different. Hence, each AS enzyme needs to be purified individually for study.

Studying Arabidopsis thaliana Using a Molecular-Genetic Approach

Arabidopsis thaliana --- the model plant

Prof. Lam used a molecular-genetic approach to explore the functions of each member of the AS gene family. He and his team began by analysing the plant Arabidopsis thaliana, which has all the characteristics of higher plants. It has a simple hereditary system, and is the first plant in the world to have been genetically decoded. The results of the genome project confirmed Prof. Lam's early findings that the AS family has a total of three genes. Prof. Lam believes that the knowledge gained from Arabidopsis thaliana will be applicable to other plants with higher agricultural and economic values.

The researchers cloned the genes in Arabidopsis thaliana (ASN1, ASN2, and ASN3) and constructed transgenic plants that overproduce these genes to study their physiological roles.

Cultivating ASN1 Overexpressing Lines

Screening for ASN1 homozygous transgenic plants. When grown on selection medium, untransformed plants turn yellow while transformed plants stay green.

Elevation of the ASN mRNA levels in ASN overexpressing lines --- L: light-grown plants; D: dark-adapted plants.

The initial focus of the research was ASN1. The researchers returned the cloned ASN1 gene to the plant to produce ASN1 overexpressing lines, in order to observe nitrogen metabolism in such lines. They discovered that under both light and dark conditions, the ASN1 gene in these transgenic lines is very active, producing high amounts of mRNA and asparagine synthetase, which subsequently causes a sharp increase in the level of free asparagine in leaves. In comparison, the ASN1 gene in the control plants expresses strongly only in the dark. This is because, in the absence of photosynthesis, the plants, to prevent wastage, catalyzes asparagine to store nitrogen temporarily.

Coinciding with Traditional Cultivation Model

Scientists have used elevated AS activities and asparagine levels in leaves as parametres to screen for high grain protein in maize and rye. One purpose of Prof. Lam's research is to identify the relationship between asparagine level in leaves and protein level in seeds.

Significant increase in seed nitrogen content in ASN1 overexpressing lines as compared to the controls. (*, P<0.05)

The researchers observed that in ASN1 overexpressing lines, there is a significant increase in the level of free asparagine not only in leaves, but also in green siliques (fruits). However, in both transgenic plants and in normal, wild-type plants, the free amino acid content in seeds is found to be much lower than that in leaves or siliques. This suggests that during seed development, nitrogen resources will be transported from leaves and fruits in the form of free asparagine, and stored as protein in seeds for future use. In ASN1 overexpressing lines, the protein content in seeds is some five to ten per cent higher as compared to the controls. This result is consistent with the traditional wisdom in seed selection for propagation.

Prof. Hon-ming Lam received his BS and M.Phil. from the Department of Biology of The Chinese University in 1985 and 1987 respectively. He then pursued research at Northwestern University, obtaining his Doctor of Philosophy in molecular biology in 1992. He worked as a research scientist at New York University prior to joining his alma mater as an assistant professor in 1997. He is also a visiting professor to Sichuan University and The Chinese Academy of Agricultural Science. Prof. Lam's current research interest is plant molecular biology, in particular, plant signal transduction, crop improvement, plant functional genomics, and using plants as bioreactors. He is now the deputy director of the Area of Excellence on Plant and Fungal Biotechnology, one of three research programmes selected for funding by the University Grants Committee among all universities in Hong Kong. Prof. Lam's research output has been published in international journals,including Nature, Proceedings of the National Academy of Sciences USA, Annual Review of Plant Physiology and Plant Molecular Biology, Plant Cell, Plant Journal, and Plant Physiology.

Implications of the Findings

This research shows that the nitrogen metabolism of a plant can be altered by the manipulation of a single gene. In particular, it shows that in ASN1 overexpressing lines, the dramatic increase in free asparagine will mean that the additional nitrogen resources are subsequently allocated to seeds. The study of Arabidopsis thaliana not only elucidates the transportation and storage of nitrogen in plants, but the results also have great implications for cereal crops. Previous physiological studies have shown that nitrogen re-allocation, especially during leaf senescence, is very important for efficient grain-filling of cereals. Prof. Lam indicated that in the future, it will be important to test whether overproduction of asparagine in ASN1 transgenic cereals will also enhance the nitrogen content of seeds in these important crop plants, and to determine whether such enhancement is at the expense of other vital metabolic components.

Prof. Lam is currently concentrating on ASN1 while beginning to develop research into ASN2 and ASN3. He predicts that the prime function of ASN2 may be to combat stressful conditions, while little is known about ASN3 due to its low level of expression.