NOD26-type aquaporins in nodules of Medicago truncatula and Medicago sativa.

FRARE, ROMINA; BOTTERO, EMILIA; STRITZLER, MARGARITA ; PASCUAN, CECILIA ; ALLEVA, KARINA; AYUB, NICOLÁS; SOTO, GABRIELA

Congreso; RAFV 2021; 2021

SAFV

Legumes are important cultivated species in part due to symbiotic nitrogen fixation by rhizobia. Medicago truncatula is the closest plant model of alfalfa. In this work, we present the genomic and functional characterization of the NOD26a and NOD26b genes of the Medicago truncatula and Alfalfa. These genes code for aquaporin proteins of the NOD26-like sub-family. With the hypothesis that these NOD26 channels are located in the symbiont nodule fulfilling functions related to nodulation, N2 fixation and / or the establishment of symbiosis, we explored the expression profile of each NOD26 in the whole plant through real-time PCR tests and it confirmed that MtNOD26a and MtNOD26b are highly expressed in nodules. In addition, the generation and evaluation of transgenic events pMtNOD26a::GUS and pMtNOD26b::GUS showed a strong GUS activity in nodules being the most intense expression in the nitrogen fixation zone. We apply different strategies to characterize the physiological role of these aquaporins. On the one hand, single mutant T-DNA insertion plants of Medicago truncatula were obtained for each aquaporin and these plants showed affected nodulation patterns, delay in nodulation, and deficiency in nitrogen fixation. In addition, we generated alfalfa mutants via CRISPR/Cas9 gene editing. Through the analysis of the HRFA profiles and sequencing of these mutants of alfalfa, we found the Knock-out for both NOD26 and we evaluated the nodulation patterns and nitrogen fixation of these mutants under conditions of limited nitrogen.