The 5′ untranslated region of the soybean cytosolic glutamine synthetase β1 gene contains prokaryotic translation initiation signals and acts as a translational enhancer in plants

Abstract

Glutamine synthetase (GS) catalyzes the synthesis of glutamine from glutamate and ammonia. In plants, it occurs as two major isoforms, a cytosolic form (GS1) and a nuclear encoded chloroplastic form. The focus of this paper is to determine the role of the 5′UTR of a GS1 gene. GS1 gene constructs with and without its 5′ and 3′ UTRs, driven by a constitutive promoter, were agroinfiltrated into tobacco leaves and the tissues were analyzed for both transgene transcript and protein accumulation. The constructs were also tested in an in vitro transcription/translation system and in Escherichia coli. Our results showed that while the 3′ UTR functioned in the destabilization of the transcript, the 5′ UTR acted as a translation enhancer in plant cells but not in the in vitro translation system. The 5′UTR of the GS1 gene when placed in front of a reporter gene (uidA), showed a 20-fold increase in the level of GUS expression in agroinfiltrated leaves when compared to the same gene construct without the 5′UTR. The 5′UTR-mediated translational enhancement is probably another step in the regulation of GS in plants. The presence of the GS1 5′ UTR in front of the GS1 coding region allowed for its translation in E. coli suggesting the commonality of the translation initiation mechanism for this gene between plants and bacteria.

Cytosolic Glutamine Synthetase Gln1;2 Is the Main Isozyme Contributing to GS1 Activity and Can Be Up-Regulated to Relieve Ammonium Toxicity

“…3C) suggests post-transcriptional regulation of GS1 transcripts in plants exposed to high ammonium. The 59-UTR of Gln-1;3 may function as a translational enhancer (Ortega et al, 2012) and may have contributed to the increase in GS1 protein in the gln1;2 and gln1;1:gln1;2 knock-out lines under the high ammonium conditions. Post-transcriptional and post-translational regulation may have been involved in maintaining the level of GS2 protein (Fig.…”

Comparison of alfalfa plants overexpressing glutamine synthetase with those overexpressing sucrose phosphate synthase demonstrates a signaling mechanism integrating carbon and nitrogen metabolism between the leaves and nodules

“…The regulation of GS 1 gene expression is not limited to transcription (Sengupta‐Gopalan & Ortega, ). It has been shown that GS 1 is regulated at the level of transcript stability, mediated by its 3′UTR (Ortega et al., ; Simon & Sengupta‐Gopalan, ) and at the translational level by the 5′UTR (Ortega, Wilson, & Sengupta‐Gopalan, ). Furthermore, GS is subject to extensive posttranslational modification like phosphorylation (Finnemann & Schjoerring, ; Lima, Seabra, Melo, Cullimore, & Carvalho, ), ubiquitination, and binding with other proteins (Seabra, Silva, & Carvalho, ).…”

Efficient expression of a heterologous gene in plants depends on the nucleotide composition of mRNA’s 5'-region

“…Currently, the 5'-UTRs and their influence on the efficiency of gene expression is being studied intensively [8,9]. Basically, comparative studies of 5'-UTRs of various genes are being conducted using reporters [10,11]. It was demonstrated in these studies that the nucleotides in the 5'-UTR, located above the start codon, significantly affect the level of translation initiation in eukaryotes, including plants [12].…”

“…Currently, elements located below the transcription start codon that can control the transcription [6] or translation [15], as well as both transcription and translation [16], were identified. However, the structure and composition, as well as the role of such regulatory sequences, controlling gene expression particularly in plants are still poorly studied [11,14]. A variety of functions and opposite effects of CpG dinucleotides in regulatory and coding regions of eukaryotic genes allows making the assumption that they may play a key role in the regulation of gene expression [6,15,17].…”