Proceedings of the XLVI Italian Society of Agricultural Genetics - SIGA Annual Congress

Giardini Naxos, Italy - 18/21 September, 2002

ISBN 88-900622-3-1

Oral Communication Abstract - S4d

TRANSFORMATION OF LOTUS CORNICULATUS PLANTS WITH E.COLI ASPARAGINE SYNTHETASE A: EFFECT ON NITROGEN ASSIMILATION AND PLANT DEVELOPMENT

Bellucci M.*, Ederli L.**, Pasqualini S.**

*) Istituto di Ricerche sul Miglioramento Genetico delle Piante Foraggere, Via Pennetti Pennella 22, 06128 Perugia, Italia

**) Dipartimento di Biologia Vegetale e Biotecnologie Agroambientali, Università di Perugia, Borgo XX Giugno 74, 06121 Perugia, Italia

amino acids, Asparagine synthetase, Lotus corniculatus, nitrogen assimialtion, floral development

Asparagine and glutamine are major forms of nitrogen in phloem sap of many higher plants. In L.japonicus asparagine can account for 86% of the nitrogen representing the principal N-source for amino acid synthesis. Asparagine synthetase (AS) catalyzes the formation of asparagine from aspartate with either glutamine or ammonia as the nitrogen source. In Escherichia coli asparagine is synthesized by the action of two distinct enzymes: AsnA, which uses ammonia as the only nitrogen donor, and AsnB, which can use both glutamine and ammonia as substrates and has a preference for glutamine. In vascular plants, glutamine-dependent AS is the primary source of asparagine. In this study, the possibility to endow plants with ammonia-dependent AS activity was investigated by heterologous expression of E. coli asnA gene with the aim of opening a new ammonium assimilation pathway in plants resulting in a stimulatory effect of growth and green mass production. The bacterial gene was placed under the control of light-dependent promoters and introduced by transformation in L.corniculatus plants. L.corniculatus was chosen as a model plant because it is a legume species (asparagine is the principal compound involved in nitrogen transport in these species) with economical importance as forage plant, and it is tractable to genetic transformation and regeneration. Putative transgenic plants were analysed by PCR and Southern blot to confirm their transgenic status and to know the asnA copy number. Surprisingly, northern blot analysis with poly (A)⁺ RNA showed that only one plant out of 22 transgenics showed an evident hybridization signal of 1.8 kb corresponding to the asnA mRNA. In order to correlate the asnA silencing with the methylation status of the T-DNA, a fine analysis of two 5’-CTGCAG-3’ Pst I sites was performed. Genomic DNA was digested with the methylation-sensitive Pst I endonuclease and analysed by PCR and Southern blot. Results show that plant pBM.asnA-11,which has a detectable steady-state level of asnA mRNA, was not methylated. The other plants, without detectable asnA mRNA, have the Pst I sites methylated. To determine whether the presence of the asnA mRNA in plant pBMasnA-11 leads to the synthesis of active enzyme, AsnA activity was measured in leaves. AsnA activity was almost not detectable in wild type plant, while the transformation induced a significant activity of AsnA. Moreover, plant pBM.asnA-11 was characterised by a premature flowering and an inhibition of growth. As concern the total free amino acids accumulation a slight, but significant reduction (-5.6 %) in pBMasnA-11 transformed plant was observed. The content of asparagine in wild type plant was about 2.5 fold compared to pBMasnA-11. The transformed plant showed glutamine concentration about 4 fold higher than that of wild type plants, while the level of aspartate was markedly smaller. The transformation with asnA also induced a significant reduction of photosynthesis, when it was measured at saturating light condition and at ambient CO₂.