Molecular and biochemical approximation to polyamine roles in tolerance mechanisms to salt stress in Lotus spp.

PAZ R, DH SÁNCHEZ, FL PIECKENSTAIN, SJ MAIALE, AI SANNAZZARO, JC CUEVAS, A CHIESA, G BONA, OA RUIZ

Chascomús, Argentina

Taller; Taller interdisciplinario sobre Lotus spp y sus simbiontes, Chascomús, Argentina; 2004

IIB-INTECh (UNSAM-CONICET)

Lotus glaber is the most important legume in the saline alkaline-lowlands of the Salado river Basin. This region (approximately 9,000,000 Ha), located in Buenos Aires Province (Argentina) is devoted to the breeding of cattle. In order to increase forage yield and improve the quality of their pastures, regional farmers utilize L. glaber, whose adaptability to saline soils is well known. The economic importance of this legume has led to an increasing number of studies regarding the physiological basis of its salt tolerance. Polyamines are aliphatic amines of low molecular weight, positively charged at physiological pH. The distribution of these positive charges permits their interaction with proteins, membrane lipids and DNA. It is well known that the activity of the plant enzymes of polyamine biosynthesis is induced under abiotic stress, including salinization. With this idea in mind, we evaluated the effect of salt stress on the polyamine pathway in L. glaber. As praline is a traditional stress marker in plants, we evaluated its levels under similar stress conditions. To understand the response of Lotus glaber to salt stress, we evaluated 15 days-old seedlings germinated and grown under 0, 25, 50 and 75 mM NaCl. We observed a progressive accumulation of sodium and potassium loss. Simultaneously, we observed an increase in praline levels and accumulation of spermine, coincidently with a decrease in spermidine levels. In addition, we analyzed a natural population of L. glaber collected from lowlands of the Salado River Basin. We isolated 103 genotypes and cloned them by nodal cuttings. Thirty-days-old genotypes were exposed to 300 mM NaCl (n=5), and average days to death were determined. A Gaussian distribution was obtained. The survival time varied from 12 to 30 days. Five genotypes of each extreme were selected and cloned and irrigated with 0 and 150 mM NaCl. Based on their differential relative growth rates, the genotypes were classified in sensitive and tolerant. We concluded that the preliminary classification based on the survival time under strong saline conditions are not representative of their tolerance conditions, because some genotypes initially considered as sensitive were later tolerant and vice-versa. Analysis of polyamine content demonstrated that spermine accumulates less in tolerant then in sensitive genotypes. Simultaneously, praline presents a progressive accumulation in both genotypes, but was lower in tolerant ones. Complementary, and taking into accont the results obtained, we generated Lotus corniculatus transgenic plants that over-express, under the control of the constitutive promoter 35SCaMV, a putative spermidine synthase gene cloned from tobacco.  The lines obtained presented onstitutive higher levels of spermidine and spermine than non-transformed controls. Moreover, we observed that these transgenic plants under saline stress showed a decrease in spermidine concentration and an increase of spermine levels, suggesting an activation of the spermine synthase activity. Under similar conditions, proline levels decreased. At the present, we are working in the generation of Lotus spp transgenic plants that potentially over-express regulatory enzymes of polyamine biosynthesis under the control of a stress-inducible promoter. This promoter, designated as RD29A, was cloned from Arabidopsis thaliana and is under evaluation in our lab. Binary vectors harbouring the arginine decarboxylase gene under the control of this promoter were successfully assayed in hairy roots of L. coniculatus, suggesting a conserved stress-signalling pathway in different species.