Prolina endógena e exógena em plantas de tabaco submetidas à deficiência hídrica: respostas fisiológicas, bioquímicas, moleculares e no perfil ionômico

Abstract

In order to mitigate the effects of environmental stresses, plants synthesize and accumulate organic solutes, called osmoprotectors, such as proline. The use of transgenic plants with a high endogenous accumulation of proline and also the exogenous application of this amino acid has been shown to benefit plants under various stresses. By acting on the redox balance, proline biosynthesis might replace/complement the energy dissipation, malate valve and alternative oxidase pathways, by regenerating the NADP+ electron acceptor, reducing the effects of stress. Also, the biochemical mechanisms involved in the accumulation of this amino acid, due to its effect in increasing the tolerance to water deficiency, might assist in the absorption of mineral elements in plants under water deficit. The objective of this study was to evaluate the physiological, biochemical, molecular and ionomic profile responses of wild and genetically modified tobacco plants for overproduction of proline submitted to exogenous applications of this osmolyte under water deficiency. The experiment was carried out in a greenhouse, using a transgenic event with constitutive expression of the VaP5CSF129A gene and the untransformed (wild) control, two exogenous proline concentrations: 0 and 10 mM and two water regimes: irrigated and deficiency water. Thirty days after transplanting the seedlings into pots, three foliar applications of proline were performed and irrigation was suspended three days after the last application for nine days. After this period, the plants were rehydrated, evaluating the recovery of these 48 h after rehydration. Physiological analyzes of relative water content, leaf gas exchange and biomass, biochemical evaluations of proline content, lipid peroxidation, total soluble protein content and total chlorophyll content were performed. The expression of the P5CS1, P5CS2, P5CR, ProDH1, ProDH2, P5CDH, NADP-MDH, NAD-MDH, AOX and VaP5CSF129A transgene, the activity of the enzymes NADP-MDH and ProDH and the ratios NADP+/NADPH and NAD+/NADH were performed. The content of N, P, K, Ca, Mg, S, B, Cu, Fe, Mn and Zn was also determined on the ninth day of irrigation suspension. Transgenic plants accumulated a higher content of proline under water deficiency and after recovery than wild plants, which was even greater with exogenous application. The higher content of proline was caused by the increase in the expression of the genes responsible for its biosynthesis, mainly NtP5CS1, and by the reduction of the expression of those related to the catabolic pathway, especially NtProDH2. Transgenic plants were less affected by the water deficit and exhibited greater recovery ability. NADP-MDH and AOX were less active in transgenic plants, mainly when exogenous application of proline was used, suggesting that this amino acid acted as a complementary/compensatory mechanism for the energy dissipation pathways. With few exceptions, the content of mineral elements was higher with proline supplementation in both irrigated and stressed plants, showing that this treatment had greater effect in the ionomic profile of tobacco plants compared to the applied water deficit.