Abstract
Here, we monitor the effects of ectopic overexpression of genes for pea asparagine synthetase (AS1) in transgenic tobacco (Nicotiana tabacum). The AS genes of pea and tobacco are normally expressed only during the dark phase of the diurnal growth cycle and specifically in phloem cells. A hybrid gene was constructed in which a pea AS1 cDNA was fused to the cauliflower mosaic virus 35S promoter. The 35S-AS1 gene was therefore ectopically expressed in all cell types in transgenic tobacco and constitutively expressed at high levels in both the light and the dark. Northern analysis demonstrated that the 35S-AS1 transgene was constitutively expressed at high levels in leaves of several independent transformants. Furthermore, amino acid analysis revealed a 10- to 100-fold increase in free asparagine in leaves of transgenic 35S-AS1 plants (construct z127) compared with controls. Plant growth analyses showed increases (although statistically insignificant) in growth phenotype during the vegetative stage of growth in 35S-AS1 transgenic lines. The 35S-AS1 construct was further modified by deletion of the glutamine-binding domain of the enzyme (gln[delta]AS1; construct z167). By analogy to animal AS, we reasoned that inhibition of glutamine-dependent AS activity might enhance the ammonia-dependent AS activity. The 3- to 19-fold increase in asparagine levels in the transgenic plants expressing gln[delta]AS1 compared with wild type suggests that the novel AS holoenzyme present in the transgenic plants (gln[delta]AS1 homodimer) has enhanced ammonia-dependent activity. These data indicate that manipulation of AS expression in transgenic plants causes an increase in nitrogen assimilation into asparagine, which in turn produces effects on plant growth and asparagine biosynthesis.
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- Bevan M. Binary Agrobacterium vectors for plant transformation. Nucleic Acids Res. 1984 Nov 26;12(22):8711–8721. doi: 10.1093/nar/12.22.8711. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dilworth M. F., Dure L. Developmental biochemistry of cotton seed embryogenesis and germination: x. Nitrogen flow from arginine to asparagine in germination. Plant Physiol. 1978 Apr;61(4):698–702. doi: 10.1104/pp.61.4.698. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Felton J., Michaelis S., Wright A. Mutations in two unlinked genes are required to produce asparagine auxotrophy in Escherichia coli. J Bacteriol. 1980 Apr;142(1):221–228. doi: 10.1128/jb.142.1.221-228.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Givan C. V., Joy K. W., Kleczkowski L. A. A decade of photorespiratory nitrogen cycling. Trends Biochem Sci. 1988 Nov;13(11):433–437. doi: 10.1016/0968-0004(88)90217-4. [DOI] [PubMed] [Google Scholar]
- Herskowitz I. Functional inactivation of genes by dominant negative mutations. Nature. 1987 Sep 17;329(6136):219–222. doi: 10.1038/329219a0. [DOI] [PubMed] [Google Scholar]
- Hongo S., Sato T. Some molecular properties of asparagine synthetase from rat liver. Biochim Biophys Acta. 1983 Feb 15;742(3):484–489. doi: 10.1016/0167-4838(83)90265-0. [DOI] [PubMed] [Google Scholar]
- Humbert R., Simoni R. D. Genetic and biomedical studies demonstrating a second gene coding for asparagine synthetase in Escherichia coli. J Bacteriol. 1980 Apr;142(1):212–220. doi: 10.1128/jb.142.1.212-220.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Joy K. W., Ireland R. J., Lea P. J. Asparagine synthesis in pea leaves, and the occurrence of an asparagine synthetase inhibitor. Plant Physiol. 1983 Sep;73(1):165–168. doi: 10.1104/pp.73.1.165. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Stulen I., Oaks A. Asparagine synthetase in corn roots. Plant Physiol. 1977 Nov;60(5):680–683. doi: 10.1104/pp.60.5.680. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tsai F. Y., Coruzzi G. Light represses transcription of asparagine synthetase genes in photosynthetic and nonphotosynthetic organs of plants. Mol Cell Biol. 1991 Oct;11(10):4966–4972. doi: 10.1128/mcb.11.10.4966. [DOI] [PMC free article] [PubMed] [Google Scholar]