The 5′UTR Intron of Arabidopsis GGT1 Aminotransferase Enhances Promoter Activity by Recruiting RNA Polymerase II

Abstract

Photorespiration is essential for the detoxification of glycolate and recycling of carbon to the Calvin Benson Bassham cycle. Enzymes participating in the pathway have been identified, and investigations now focus on the regulation of photorespiration by transporters and metabolites. However, regulation of photorespiration on the gene level has not been intensively studied. Here, we show that maximum transcript abundance of Glu:glyoxylate aminotransferase 1 (GGT1) is regulated by intron-mediated enhancement (IME) of the 59 leader intron rather than by regulatory elements in the 59 upstream region. The intron is rich in CT-stretches and contains the motif TGTGATTTG that is highly similar to the IME-related motif TTNGATYTG. The GGT1 intron also confers leaf-specific expression of foreign promoters. Quantitative PCR analysis and GUS activity measurements revealed that IME of the GGT1 59UTR intron is controlled on the transcriptional level. IME by the GGT1 59UTR intron was at least 2-fold. Chromatin immunoprecipitation experiments showed that the abundance of RNA polymerase II binding to the intron-less construct is reduced.

Intron-Mediated Enhancement: A Tool for Heterologous Gene Expression in Plants?

“…The fact that the 5′UTR intron of GGT1 is able to drive leaf expression of both GGT2 and GLDP1-P2 as well as expression in trichomes of a root-specific PEROXIDASE (Laxa et al, 2016) might support the theory that expression is enhanced in a tissue in which expression is already determined but below the detection limit. Supporting this theory, Rose and Last (1997) showed that an increase in mRNA accumulation is depending on the presence of the AtPAT1 intron, while there was no change in the rate of transcription when compared to the intronless construct (Rose and Last, 1997).…”

“…In general, the impact of IME on transcription does not exceed two- to threefold (Rethmeier et al, 1997; Rose and Last, 1997; Furger et al, 2002; Samadder et al, 2008; Moabbi et al, 2012; Laxa et al, 2016). Thus, a significant difference in transcription that is mediated by an intron compared to an intronless control is even harder to detect than an increase in mRNA accumulation by 20-fold or more.…”

“…The presence of the two first introns of PAT1 enhanced transcription of gusA below twofold but both mRNA accumulation and GUS activity by 30-fold in stably transformed Arabidopsis plants. The 5′UTR intron of glutamate:glyoxylate aminotransferase 1 ( GGT1 ) enhanced transcription as indicated by a decrease in RNA polymerase II binding in the absence of the 5′UTR intron from a chimeric gusA construct (Laxa et al, 2016). In contrast to the above mentioned studies no further enhancement was seen on either mRNA or GUS activity level.…”

“…Supporting this theory, Rose and Last (1997) showed that an increase in mRNA accumulation is depending on the presence of the AtPAT1 intron, while there was no change in the rate of transcription when compared to the intronless construct (Rose and Last, 1997). On the other hand, the presence of the intron had a direct influence on RNA polymerase II binding to the chimeric GGT1 :: gusA construct indicating a recruitment of the transcriptional machinery to the promoter (Laxa et al, 2016). …”

“…In many publications not even the number of individual transformation events is evident. However, the variation between either transformation events or different experiments can be quite high as seen for transgenic lines (Snowden et al, 1996; Chung et al, 2006; Laxa et al, 2016). Sometimes results are difficult to judge when only specific tissues are affected by the presence of an intron (Giani et al, 2009; Karthikeyan et al, 2009).…”