Alternative mRNA splicing for the control of fumarase activity in Arabidopsis

ANDREO CS; DRINCOVICH, M. F.; ZUBIMENDI JP; TRONCONI MA

Ciudad Autónoma de Buenos Aires

Congreso; Reunión Conjunta de Sociedades de Biociencias; 2017

Arabidopsis thaliana possesses two fumarase genes (FUM), At-FUM1 (At2g47510) encoding for the mitochondrial Krebs cycle-associatedenzyme and AtFUM2 (At5g50950) for a cytosolic isoformrequired for the massive accumulation of fumarate. While AtFUM1codifies a single transcript, AtFUM2 could codify two different isoenzymes(AtFUM2 and AtFUM2b) through an alternative maturationprocess of the primary transcript. The characterization of recombinantforms of these proteins indicated that AtFUM2b lacks ofenzymatic activity due to absence of the amino acid residues thatare encoded by the last exon, as the AtFUM2b transcript retainsan intron containing a stop codon. Here, we further analyzed thephysiological role of this AtFUM2-derived aberrant mRNA. In orderto determinate whether it is translated in vivo, the AtFUM2b cDNAwas cloned in frame with fluorescent green protein (GFP) codingsequence into a binary vector for the transient Agrobacterium-mediatedtransformation of Nicotiana benthamiana leaves. The resultsindicated that, despite AtFUM2b transcript was present, no fluorescencewas detected in infiltrated Nicotiana leaves, noting that theprotein is not translated in vivo and pointing out a regulatory rolefor the not fully processed AtFUM2b mRNA into the cell. On theother hand, the abundance of AtFUM1, AtFUM2 and AtFUM2b transcriptsin Arabidopsis plants grown under different conditions wasquantified through RT-qPCR. In general, the three mRNA were differentiallyaccumulated under the assayed conditions of biotic andabiotic stress, and with the AtFUM2 gene expressed to a greaterextent than the AtFUM1. Particularly, the AtFUM2b abundance was significantly lower than AtFUM2 and, in several conditions, bothAtFUM2-derived transcripts accumulated inversely. Overall, our resultsindicate that in Arabidopsis the FUM activity is regulated atthe level of RNA-processing for control of the C4-organic acids fluxthrough metabolism.