Characterization of Heterologous Multigene Operons in Transgenic Chloroplasts. Transcription, Processing, and Translation

Abstract

The first characterization of transcriptional, posttranscriptional, and translational processes of heterologous operons expressed via the tobacco (Nicotiana tabacum) chloroplast genome is reported here. Northern-blot analyses performed on chloroplast transgenic lines harboring seven different heterologous operons revealed that polycistronic mRNA was the predominant transcript produced. Despite the lack of processing of such polycistrons, large amounts of foreign protein accumulation was observed in these transgenic lines, indicating abundant translation of polycistrons. This is supported by polysome fractionation assays, which allowed detection of polycistronic RNA in lower fractions of the sucrose gradients. These results show that the chloroplast posttranscriptional machinery can indeed detect and translate multigenic sequences that are not of chloroplast origin. In contrast to native transcripts, processed and unprocessed heterologous polycistrons were stable, even in the absence of 3# untranslated regions (UTRs). Unlike native 5#UTRs, heterologous secondary structures or 5#UTRs showed efficient translational enhancement independent of cellular control. Abundant read-through transcripts were observed in the presence of chloroplast 3#UTRs but they were efficiently processed at introns present within the native operon. Heterologous genes regulated by the psbA (the photosystem II polypeptide D1) promoter, 5# and 3#UTRs have greater abundance of transcripts than the endogenous psbA gene because transgenes were integrated into the inverted repeat region. Addressing questions about polycistrons, and the sequences required for their processing and transcript stability, are essential in chloroplast metabolic engineering. Knowledge of such factors would enable engineering of foreign pathways independent of the chloroplast complex posttranscriptional regulatory machinery.Plastid genes in higher plants are mainly organized as operons, of which more than 60 have been described in the tobacco (Nicotiana tabacum) chloroplast genome (Sugita and Sugiura, 1996). These may group genes of related or unrelated functions, the former being the most common (Barkan, 1988;Rochaix, 1996). Most of these genes are transcribed into polycistronic precursors that may be later processed and modified to render the transcripts competent for translation (Eibl et al., 1999;Barkan and Goldschmidt-Clermont, 2000;Monde et al., 2000b).The processing mechanisms for translation regulation in chloroplast genes of higher plants are still largely unknown. The general consensus is that most native primary transcripts require processing to be functional (Barkan, 1988;Zerges, 2000;Meierhoff et al., 2003) and that posttranscriptional RNA processing of primary transcripts represents an important control of chloroplast gene expression (Hashimoto et al., 2003;Nickelsen, 2003). However, it is believed that more than one pathway may be involved in transcript processing (Danon, 1997;Choquet and Wollman, 2002).For example, several studies have shown that the regul...

Codon Optimization to Enhance Expression Yields Insights into Chloroplast Translation

“…Likewise, details of the homologous recombination process and the deletion of mismatched nucleotides were evident using heterologous flanking sequences (Ruhlman et al, 2010). The translation of native polycistrons without the need for processing to monocistrons has been demonstrated (Barkan, 1988;Zoschke and Barkan, 2015), but the similarity of this process using heterologous polycistrons engineered via the chloroplast genome offered even more direct evidence for this process (De Cosa et al, 2001;Quesada-Vargas et al, 2005). The insertion of replication origins into chloroplast vectors offered further insight into minimal sequences required to study this process (Daniell et al, 1990).…”

Complete plastid genome sequence of the chickpea (Cicer arietinum) and the phylogenetic distribution of rps12 and clpP intron losses among legumes (Leguminosae)

“…Therefore, complete plastid genome sequences of several major crop species including fiber crops , tubers (Daniell et al, , 2008, cereals (Saski et al, 2007), trees (Steane, 2005;Bausher et al, 2006;Ravi et al, 2006;Samson et al, 2007), vegetables (Ruhlman et al, 2006), fruits Daniell et al, 2006) and legumes (Saski et al, 2005;Guo et al, 2007) have been determined recently. Plastid genetic engineering offers a number of unique advantages including high level of transgene expression (DeCosa et al, 2001), multi-gene engineering in a single transformation event (Quesda-Vargas et al, 2005), transgene containment via maternal inheritance (Daniell, 2002;Daniell, 2007) or cytoplasmic male sterility . Plastid transgenic lines also lack gene silencing (DeCosa et al, 2001;Lee et al, 2003), position effect due to site specific transgene integration and pleiotropic effects due to subcellular compartmentalization of transgene products (Lee et al, 2003;Daniell et al, 2001;Leelavathi et al, 2003); concerns of transgene silencing, position effect and pleiotropic effects are often encountered in nuclear genetic engineering.…”

The complete nucleotide sequence of the cassava (Manihot esculenta) chloroplast genome and the evolution of atpF in Malpighiales: RNA editing and multiple losses of a group II intron

“…Chloroplast genetic engineering would be ideal to address this problem because of the high-dosage strategy that can kill Bacillus thuringiensis susceptible and resistant insects (Kota et al 1999;DeCosa et al 2001). In addition, multi-gene engineering (DeCosa et al 2001;Quesada-Vargas et al 2005) should facilitate the introduction of genes that code for insecticidal proteins. Several other agronomic traits engineered via the chloroplast genome including disease resistance (DeGray et al 2001), salt tolerance (Kumar et al 2004a), drought tolerance (Lee et al 2003), and herbicide resistance (Daniell et al 1998;Dufourmantel et al 2007), are valuable for cassava biotechnology.…”