The overexpression of asparagine synthetase A from E. coli affects the nitrogen status in leaves of lettuce (Lactuca sativa L.) and enhances vegetative growth
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The asparagine synthetase A (EC 220.127.116.11) of E. coli (AS-A) mainly uses ammonia to produce asparagine, a key nitrogen transporter in plants. The AS-A encoding gene (asnA) was expressed constitutively in lettuce cultivar ‘Cortina’ under the control of pMAC, a chimerical promoter, to induce phenotypical alterations of plant growth and quality as a consequence of nitrogen status changes. Nine fertile transgenic lines harbouring independent T-DNA insertions were recovered. Primary transformants shared new visible traits such as a higher leaf number and wider leaf surface than the wild-type. The progeny of three primary transformants stably maintained these phenotypes, to which the synthesis of both asnA transcript and protein were associated. In pMAC:asnA plants, seed germination, formation and development of leaves, bolting and flowering occurred earlier than non-transformed plants. Twenty-eight days after sowing (das), transgenic plants showed a ca. 1.3 increase of leaf area and dry weight as compared to the wild-type. Moreover, the contents of asparagine, aspartic acid and glutamine, but not that of glutamic acid, of pMAC:asnA young plants (21 das) were greater than the wild-type. The level of total soluble protein was higher in transgenic than in non-transformed leaves borne on plants at 35, 50 and 75 das. A decrease of nitrate was also measured in pMAC:asnA leaves with respect to non-transformed ‘Cortina’, in transgenic populations at 60 das. In pMAC:asnA genotypes, the altered content of nitrogen transport amino acids, the tolerance to increasing doses of ammonium and phosphinothricin indirectly proved the AS-A enzymatic activity in lettuce.
KeywordsAsparagine synthetase A Lettuce Growth enhancement
Days after sowing
We are grateful to: Dr. Frank Schepers (Advanta Research, NTH) for providing AS-A antibodies and helpful suggestions in conducting Western blots; Dr. Carla Ticconi (University of Davis, CA, US) for criticism; Mr. Luigi Santini (IBBA-CNR) for lettuce care. This work was funded by Project EC-AIR CT no. 92–250, “Lettuce for the next century”, and by the CNR.
- Donn G, Eckes P, Müllner H, Dudits D, Feher A, Paulovics K (2002) Process for the production of plants with enhanced growth characteristics. United States Patent Application No. 20020035740Google Scholar
- Dudits D, Paulovics K, Kalmann K, Gyorgyey J, Nagy F, Bako L, Horvarth G, Eckes P, Donn G (1991) Transgenic plants expressing a prokaryotic ammonium dependent asparagine synthetase. PCT-International Patent Application WO91/11524Google Scholar
- Frugis G, Giannino D, Mele G, Nicolodi C, Chiappetta A, Bitonti MB, Innocenti AM, Dewitte W, Van Onckelen H, Mariotti D (2001) Overexpression of KNAT1 in lettuce shifts leaf determinate growth to a shoot-like indeterminate growth associated with an accumulation of isopentenyl-type cytokinins. Plant Physiol 4:1370–1380CrossRefGoogle Scholar
- Porra RJ, Thompson WA, Kriedemann PE (1989) Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. Biochim Biophys Acta 975:384–394CrossRefGoogle Scholar
- Santamaria P, Elia A, Gonnella M (1997) Changes in nitrate accumulation and growth of endive plants during the light period as affected by nitrogen level and form. J Plant Nutr 20:1255–1266Google Scholar
- Vincent R, Fraisier V, Chaillou S, Limami MA, Deleens E, Phillipson B, Douat C, Boutin JP, Hirel B (1997) Overexpression of a soybean gene encoding cytosolic glutamine synthetase in shoots of transgenic Lotus corniculatus L. plants triggers changes in ammonium assimilation and plant development. Planta 4:587–595Google Scholar