Characterization of maize spermine synthase 1 (ZmSPMS1): evidence for dimerization and intracellular location

[EN] Polyamines are ubiquitous positively charged metabolites that play an important role in wide fundamental cellular processes; because of their importance, the homeostasis of these amines is tightly regulated. Spermine synthase catalyzes the formation of polyamine spermine, which is necessary for growth and development in higher eukaryotes. Previously, we reported a stress inducible spermine synthase 1 (ZmSPMS1) gene from maize. The ZmSPMS1 enzyme differs from their dicot orthologous by a C-terminal extension, which contains a degradation PEST sequence involved in its turnover. Herein, we demonstrate that ZmSPMS1 protein interacts with itself in split yeast two-hybrid (Y2H) assays. A Bimolecular Fluorescence Complementation (BiFC) assay revealed that ZmSPMS1 homodimer has a cytoplasmic localization. In order to gain a better understanding about ZmSPMS1 interaction, two deletion constructs of ZmSPMS1 protein were obtained. The Delta N-ZmSPMS1 version, where the first 74 N-terminal amino acids were eliminated, showed reduced capability of dimer formation, whereas the Delta C-ZmSPMS1 version, lacking the last 40 C-terminal residues, dramatically abated the ZmSPMS1-ZmSPMS1 protein interaction. Recombinant protein expression in Escherichia coli of ZmSPMS1 derived versions revealed that deletion of its N-terminal domain affected the spermine biosynthesis, whereas C-terminal ZmSPMS1 truncated version fail to generate this polyamine. These data suggest that N- and C-terminal domains of ZmSPMS1 play a role in a functional homodimer. (C) 2015 Elsevier Masson SAS. All rights reserved.This work was supported by the CONACYT (Investigacion Ciencia Basica CB-2013-221075, Fortalecimiento de infraestructura INFR-2014-01-224800, Renovacion de Infraestructura INFR-2014-01224220) funding to JFJB, and funding from the Spanish MICINN/MINECO (BIO2011-23828) to AF and JC. The authors acknowledge to MC Guillermo Vidriales Escobar from IPICYT for his technical assistance in HPLC analyses.Maruri-López, I.; Hernández-Sánchez, I.; Ferrando Monleón, AR.; Carbonell Gisbert, J.; Jimenez-Bremont, J. (2015). Characterization of maize spermine synthase 1 (ZmSPMS1): evidence for dimerization and intracellular location. Plant Physiology and Biochemistry. 97:264-271. https://doi.org/10.1016/j.plaphy.2015.10.017S2642719

Expression of Escherichia coli heat-labile enterotoxin b subunit (LTB) in carrot (Daucus carota L.)

We expressed the B subunit of enterotoxigenic Escherichia coli heat-labile enterotoxin (LTB) encoded by a synthetic codon-optimized gene in carrot. An Agrobacterium-mediated transformation method was used. Thirty independent transgenic lines were regenerated via somatic embryogenesis after 6 months in culture and were transferred to a greenhouse. GM1-ELISA assay was used to assess LTB protein content in mature taproots. Some transgenic lines expressed LTB up to 0.3% of the total soluble protein, which is tenfold higher than the expression levels reported earlier using the native bacterial gene in plants. Immunological assay confirmed proper assembly of the pentameric complex and in vitro activity of the recombinant LTB protein, suggesting that it can be functional in prevention of diarrhea