Indução de tolerância ao estresse salino em plântulas de arroz por meio do pré-tratamento químico de sementes com tunicamicina

Abstract

Salinity is an abiotic fator that represents a major problem for plants, affecting several biochemical and physiological processes in crops of interest, therefore decreasing their biomasses and yield, and the increase of salinization processes in arable lands makes it a concerning issue. It is also known that salt stress induces secondary stresses, such as the oxidative one (characterized by an overproduction of reactive oxygen species) and the endoplasmic reticulum (ER) one, which is represented by an accumulation of unfolded or misfolded proteins in the ER lumen. Both can lead to programmed cell death, so strategies that are able to overcome them prove to be necessary. Chemical seed priming is a good alternative to lead to plant tolerance to abiotic stresses, for it activates defense mechanisms in

the seed, such as activation of antioxidante enzymes and positive modulation of stress- responsive metabolites in crops of importance, such as rice (Oryza sativa L.). Based on that,

this work had as goal to test the hypothesis that tunicamycin (TM), a natural antibiotic and a specific ER stressor, is capable of alleviating salt toxicity during the establishment of rice seedlings, cv. SCSBRS 113, through activation of an efficient antioxidant system, Na+ exclusion of the cell and positively modulating primary and salt-responsive metabolites. This PhD thesis was divided in two chapters: the first one was a theorical foundation about the main topics of this work. The second one, in the form of a paper, to be submitted, describes two experiments, both with completely randomized designs. The first one comprised three screenings, in order to determine the best NaCl and TM concentrations for further analyses, with increasing NaCl concentrations (0, 100, 150, 200, and 250 mM NaCl) and fixed TM concentrations (either 0 or 0.50 μg . mL-1

, screenings I and II, respectively), and the third screening was performed with increasing TM concentrations (0, 0.125, 0.250, 0.375 and 0.500 μg. mL-1

TM) and one NaCl concentration (150 mM); the second experiment comprised further analyses with four treatments, a set of assays such as contents of photosynthetic pigments (chlorophyll a, chlorophyll b, total chlorophyll and carotenoids), inorganic ions (K+ ,

Na+ , and Cl- ), and ROS (H2O2 and •O2 - ) as well as Na+

accumulation in tissues by confocal microscopy, electrolyte leakage, osmotic potential, lipid peroxidation, antioxidant enzyme activities (such as SOD, CAT, APX and GPOD) and metabolomics of shots and roots of 10 DAT (days after treatment) rice seedlings, being chosen from the screenings. Results showed that 150 mM NaCl allowed seedling establishment while 0.25 μg.mL TM was able to alleviate salinity effects in germination and biomass parameters. Moreover, TM primed seedlings, compared to unprimed ones under salinity, not only displayed increased dry mass, length, photosynthetic pigments and K+

contents, osmotic potential and antioxidant enzyme activities,

but also showed decreased electrolyte leakage, Na+

and Clcontents, ROS levels and lipid peroxidation. Metabolomics analyses revealed 40 identified metabolites, being 15 of them amino acids, 10 organic acids, 11 carbohydrates, and 4 others. TM-priming, in shoots, also modulated positively salt-defensive metabolites, such as the osmoprotectants beta-alanine and

maltose, whereas in roots it had a higher accumulation of phosphoric acid, the amino acids o- acetylserine and N-acetylserine, responsible for Fe and S metabolisms in the cell such as

energy synthesis, and also the sugars maltose, raffinose and sorbitol, which not only provide energy to the cell but also act as osmoprotectants, which might have led to a mitigation of salt effects. In conclusion, TM seed priming alleviated severe salt stress in rice seedlings by mainly activating antioxidant enzymes and also positively modulating defense metabolites that helped generate energy, sending Na+

ions out of the cell, therefore proving to be an efficient strategy to mitigate salt stress. This work provides insights that can lead to further analyses in order to better comprehend the molecular mechanisms activated by TM that lead to salt acclimation.