Caracterização dos transportadores OsNTR1.1A, OsNTR1.1B e OsNTR1.1C por meio da reversão fenotípica do mutante chl1-5 de Arabidopsis thaliana

Abstract

Nitrogen (N) uptake by plants is a key step for N use efficiency, affecting fresh mass production and yield of grains. The NRT1.1 transceptor of Arabidopsis thaliana was identified as a molecular signal of nitrate uptake (NO3-). In rice, three orthologs of the NRT1.1 named OsNRT1.1A, OsNRT1.1B and OsNRT1.1C, were identified. The objective of this work was to evaluate the overexpression of the genes OsNRT1.1A, OsNRT1.1B and OsNRT1.1C in Arabidopsis thaliana chl1-5 mutant plants to restore the transport and signaling capacity of the nitrate lost in the knockout mutant. The transformation process of A. thaliana plants was obtained by floral immersion with strains of Agrobacterium tumefaciens of lineage LBA4404 by the following constructs 35S: OsNRT1.1A: HA, 35S: OsNRT1.1B: HA, 35S: OsNRT1. 1C: HA and 35S: OsNRT1.1sa: HA (promoter: gene: HA tag). Subsequently, the antibiotic kanamycin was used to obtain segregant lineages of the transformation product, being only plants with two copies of the gene was selected for testing the differents levels of gene expressing. To verify the resistance of the mutant plants to the chlorate (NaClO3), the experiment was set up with homozygous plants, and their seeds were germinated on commercial substrate and vermiculite. At 23 days after planting, applications with 12 mM NaClO3 were started. To evaluate altered gene expression by introduction of OsNRT1.1A, OsNRT1.1B, OsNRT1.1C, primers were designed for expression analysis of the high and low affinity NO3- transporters. Two lines of each transformation were used in the experiments, including wild type plants (WT) and chl1-5 mutant plants. The chlorate test showed the ability of OsNRT1.1A, OsNRT1.1B or OsNRT1.1C to nitrate uptake, evidenced by the decrease in fresh mass caused by the reduction of chlorate to chlorite by nitrate reductase, the chlorite a toxic product to cells. The insertion of transporter OsNRT1.1B caused the largest growth reduction in the chlorate test compared to OsNRT1.1A and OsNRT1.1C, aproximating to the same levels of wild-type (WT). Expression analyzes showed that the insertion of OsNRT1.1A, OsNRT1.1B and OsNRT1.1C genes into Arabidopsis thaliana chl1-5 was able to induce the expression of OsNRT2.1 and OsNAR2.1 genes, and the alternative splicing form of OsNRT1 .1A (OsNRT1.1Asa) did not significantly affect the expression of OsNRT2.1 and OsNAR2.1. The results show the ability of orthologs of NRT1.1 in rice (OsNRT1.1A, OsNRT1.1B and OsNRT1.1C) to nitrate uptake and signal to the expression of other nitrate transporters (transceptor), which may affect the nitrogen efficiency and uptake.