Use of transformed tobacco plants with endogenously altered polyamine pools for studying the role of these molecules on the regulation of arbuscular mycorrhizal infection

ANA BERNARDINA MENENDEZ

PINAMAR

Congreso; XLI Reunión SAIB; 2005

The endosymbioses formed with arbuscular mycorrhizal fungi (AMF) plays an important role in agriculture and natural ecosystems. A number of regulatory mechanisms of plant defence response have been described during the establishment of the arbuscular mycorrhizal (AM) symbiosis including elicitor degradation, modulation of second messenger concentration, nutritional and hormonal plant defence regulation, and activation of regulatory symbiotic gene expression. Polyamines putrescine (Put), spermidine (Spd) and spermine (Spm) are aliphatic polications playing a regulatory role in growth and development. In this work , we tested an experimental design including the use of transformed tobacco plants with endogenously altered polyamine pools, for studying the putative role of these polycations on the regulation of AM infection.ucon Materials and Methods Transgenic tobacco plants transformed with pBin-HYG-TXADC, containing the Avena sativa L. (oat) arginine decarboxylase (ADC) open reading frame under the control of an inducible promoter that can be activated by addition of tetracycline were used. Plants expressing the tetracyline repressor protein were used as a control. Transgenic and control plants were germinated in a glasshouse in pots containing vermiculite : perlite (1 : 1). Plants were fed with Hewitt nutrient solution and after 6 weeks, half of the plants were treated with tetracycline. To induce the expression of the transgene, 2 mg l-1 of tetracycline were applied to the nutrient solution every 3 days. After a 4 weeks induction, half of the plants in each treatment were inoculated with Glomus intraradices Schenk and Smith. Inoculum consisted of a small spoon of chopped, mycorrhized Zea maize roots. Two weeks after inoculation, plants were harvested and processed. Free polyamines content of leaves were measured. The presence of G. intraradices  and percentage of mycorrhizal root colonization were determined visually by clearing and staining the roots with Trypan blue in lactophenol..  There were no differences in mycorrhization parameters between Tet R plants induced or not with tetracycline, whereas oat ADC plants, showed higher levels of mycorrhizal root length, entry points and vesicles due to tetracycline induction. However there were not significant correlations between these parameters and the contents of any free polyamine. Tetracycline induction decreased free spermine levels in nonmycorrhized plants transformed with the oat ADC gene, and increased free putrescine levels in mycorrhized plants transformed with the oat ADC gene. In turn, mycorrhization increased free spermine levels in non-induced TetR plants and in oat ADC induced plants. Free putrecine levels were decreased by mycorrhization in oat ADC noninduced plants. There were no differences in free spermidine levels between treatments.