Avaliação da expressão dos genes OsNRT1.1 (A, B e C) e efeito do silenciamento das isoformas OsA2 e OsA7 de PM H+-ATPases na absorção de nitrogênio em arroz

Abstract

The soil nitrogen (N) availability is generally low, leading to frequently input of fertilizer and the search of varieties with higher nitrogen use efficiency. Among the favorable parameters include higher expression of plasma membrane pumps protons and high-affinity nitrate transporters under low N cultivation. This work aimed to identify the expression pattern of OsNRT1.1A, OsNRT1.1B and OsNRT1.1C, possible orthologs of the Arabidopsis double affinity nitrate tranporter NRT1.1 (CHL1; NPF8.9) in response to NO3- and NH4+, as well as evaluate the importance of OsA2 and OsA7 proton pumps genes to N uptake. To evaluate OsNRT1.1 (A, B and C) gene expression, two experiments were performed: the first using Nipponbare rice variety and the second using IAC-47 (improved) and Piauí (Landrace) rice varieties. The OsA2 and OsA7 gene silencing was made using the artificial micro RNA (amiRNA) followed by rice genetic transformation. The experiment to evaluate the contribution of plasma membrane H+-ATPase (PM H+-ATPase) to low NO3- levels was made using IAC-47 and Piauí rice varieties. The OsNRT1.1 gene expression analysis in the roots showed that OsNRT1.1A and OsNRT1.1B are highly expressed, while OsNRT1.1C showed low expression rate. OsNRT1.1B expression was downregulated with ressuply of 2.0 mM of NO3--N, in the plants cultivated using NH4+ and N starving plants, however, OsNRT1.1B expression was upregulated with constant supply of 2.0 mM of NO3--N. After rice genetic transformation, Real Time PCR analysis showed that only one amiRNA successfully downregulated the target, furthermore the lineages silencing the target were multiplied in greenhouse. The rice lineages downregulating OsA2 (osa2.2, osa2.3, osa2.4), OsA7 (osa7.1, osa7.2, osa7.3) and control plants (plants transformed with empty IRS) were cultivated using four treatments: three days N starvation, constant with 0.2 mM of NO3--N, ressuply with 0.2 mM of NO3--N and constant with 2.0 mM of NO3--N. The osa7 mutants shown lower growth, meanwhile osa2 growth normally compared to control plants (IRS). Both mutants shown lower NO3- uptake when grown with constant 0.2 mM of NO3--N compared to control IRS plants, meanwhile the other treatments did not show differences of NO3- uptake. The PM H+-ATPase activity was lower in the osa7 and osa2 mutants compared to IRS plants in all treatments used. The V-H+-ATPase activity did not show difference in the mutants osa7 and osa2, however, V-H+-PPase activity was slower only in the osa7 mutants compared to IRS control plants. The mutant plants osa2 showed lower N uptake Efficiency index, accumulating lower total-N when fed with 0.2 mM of NO3--N constant. The results highlight that there is differentiated contribution of specific PM H+-ATPase isoforms to rice growth and to NO3- uptake efficiency.