Associação da expressão de dois genes da família MATE com a tolerância ao alumínio em três espécies de Eucalyptus sp.

Abstract

Eucalyptus is a crop of great importance to the market, as its processed wood supplies various production chains, such as paper and cellulose, charcoal and energy, and furniture. However, some species are affected by high concentrations of free aluminum present in the soil, reducing productivity. One of the main soil limitations is the presence of toxic aluminum, which becomes available to plants in soils with a pH below 5. In the Midwest, Southeast, and South regions of Brazil, where most eucalyptus plantations are located, this problem is common due to soil acidity. There is a family of genes called Multidrug and Toxic Compound Extrusion (MATE), which includes a group of proteins responsible for the exudation of organic acids in the roots. These proteins can release organic acids, such as malate and citrate, which can chelate phytotoxic aluminum and reduce its negative effects on plants. Given this, the aim of this study was to investigate and demonstrate the relationship between two genes from the MATE family and citrate exudation in three species of eucalyptus with different levels of tolerance to Al3+. For this purpose, seeds were germinated in vitro, and after 60 days, the seedlings of Eucalyptus grandis, Eucalyptus urophylla, and Eucalyptus dunnii were exposed to liquid media containing 2mM of Al3+ to stimulate citrate production and the activation of genes associated with this physiological response. Samples of the liquid medium were collected for analysis by High Performance Liquid Chromatography - Diode Array Detector (HPLC-DAD) and the enzymatic method for citrate detection using the Sigma-Aldrich® MAK333 Kit. The roots were collected, and total RNA was extracted, followed by DNase treatment to remove genomic DNA, cDNA synthesis, and primer efficiency testing for qPCR. Due to specific issues during the experiment, such as not being able to extract RNA with good quality indices, qPCR analysis, which would demonstrate gene expression under stressful conditions, was not possible. The enzymatic method for quantifying citrate release in the medium failed due to some interference from the medium where the explants were, such as pH or some component, Al3+ for example. However, the HPLC method worked, with some obstacles, to detect citrate concentrations in the medium, but the results did not show statistical variation. Therefore, it was not possible to prove the association of the candidate genes from the MATE family with Al3+ tolerance. However, the main points to be observed when conducting similar research were listed to avoid the same problems.