Proceedings of the XLVII Italian
Society of Agricultural Genetics - SIGA Annual Congress
Verona,
Italy - 24/27 September, 2003
ISBN 88-900622-4-X
Poster
Abstract - 2.55
MOLECULAR AND PHYSIOLOGICAL CHARACTERISATION OF LACTUCA
SATIVA EXPRESSING THE ASPARAGINE SYNTHETASE TYPE A OF E. COLI
D. GIANNINO, C.
NICOLODI, G. TESTONE, E. TRASARTI, L. SANTINI, D. MARIOTTI
Istituto di
Biologia e Biotecnologie Agrarie del Consiglio Nazionale delle Ricerche,
Sezione Territoriale di Roma, Via Salaria Km. 29,300, 00016 Monterotondo Scalo
(Roma)
nitrogen
metabolism, genetic transformation, asparagine synthetase A, lettuce
Plant uptake of
nitrogen implies the biochemical reduction of nitrates to ammonium, rapidly
incorporated into glutamate (Glu) and glutamine (Gln) by the glutamate and
glutamine synthases, respectively. The latter also represents the major
scavenger of ammonia deriving from nitrate reduction, amino acid degradation
and photorespiration. In plants Gln is the nitrogen donor to aspartic acid
(Asp) for the asparagine (Asn) synthesis, catalysed by the asparagine
synthetase (AS). The latter is inhibited by light, has a high specificity for
glutamine and rarely uses ammonium as substrate. Contrary to plants, in
prokaryotes the asparagine synthetase type A (asnA) transfers ammonium directly
to aspartic acid to produce apsaragine and it is not regulated by light. The
genetic transfer of the asnA gene into lettuce was attempted to
enhance nitrogen metabolism and to diminish nitrate content, a serious limit
for the product quality of this crop. Three cultivars (Cortina, Clinton and
Luxor) were transformed with asnA driven by a
35S-derived promoter (pMAC) by A. tumefaciens
Transformation efficiency was 70% and the events of single insertion of
transgene were 30%. One out of several Cortina primary transformed lines
exhibited altered phenotypes with respect to controls, thus it was selected and
selfed to reach stabilised transgene expression and homozygosity. Plants
harboring pMAC:asnA exhibited a higher growth speed, anticipation of
bolting and flowering with respect to wild type Cortina. In greenhouse winter
and summer cycles, transformed genotypes featured 1.3-1.4 fold higher fresh and
dry weights than controls, due to higher leaf number and average area. The
content of Asn, Asp and Gln of pMAC:asnA plants was 2.6 to
4.6 fold higher than controls, whereas Glu variation was not significant. In
vitro experiments showed that pMAC:asnA
genotypes were more tolerant to increasing ammonium concentrations than
controls. Nitrate content of transformed plants is currently being measured and
data will be presented and discussed.