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Expression of a chimeric uidA gene indicates that polycistronic mRNAs are efficiently translated in tobacco plastids
Abstract
Polycistronic mRNAs are the predominant form of plastid primary transcripts. To determine if there is internal initiation of translation of promoter-distal open reading frames, a promoterless uidA reporter gene was integrated into the tobacco plastid genome downstream of the rbcL gene. Monocistronic uidA mRNA does not accumulate from the promoterless uidA construct. However, due to inefficient rbcL transcription termination, a polycistronic transcription unit is created that contains the uidA gene as the second cistron. Numerous stop codons in all three reading frames between the rbcL and uidA coding regions ensure that translation of uidA initiates only from the correct start codon. The encoded reporter gene product, beta-glucuronidase (GUS) accumulates to high levels in the transplastomic plants indicating that promoter-distal cistrons can be efficiently translated in plastids.
... The direction and the expected size of psbC:uidA transcripts and the location of the relevant restriction enzyme sites when stably integrated into the tobacco genome are shown in Fig. 1B www.nature.com/scientificreports/ expected ~ 2.1 kb major band representing uidA transcript 28 , the presence of a minor band of lower size was also observed in all transplastomic lanes, which might represent the uidA transcripts that are not denatured completely at the time of gel loading. However, uidA transcript levels were significantly less where CUG was used as the start codon. ...
... For this purpose, we chose the promoter of tobacco psbC gene having GUG as the native start codon 35 and another promoter of rice psbA gene with AUG as the native start codon 30 www.nature.com/scientificreports/ other than the desired locus (rbcL-accD), that may, leading to large DNA deletions and rearrangements in the plastid genome 28 . An important finding in the present study is the very low level of transcription of uidA under the psbC promoter with CUG as the start codon (Fig. 2C) as compared to other start codons (AUG, GUG or UUG) tested. ...
Chloroplasts have evolved from photosynthetic cyanobacteria-like progenitors through endosymbiosis. The chloroplasts of present-day land plants have their own transcription and translation systems that show several similarities with prokaryotic organisms. A remarkable feature of the chloroplast translation system is the use of non-AUG start codons in the protein synthesis of certain genes that are evolutionarily conserved from Algae to angiosperms. However, the biological significance of such use of non-AUG codons is not fully understood. The present study was undertaken to unravel the significance of non-AUG start codons in vivo using the chloroplast genetic engineering approach. For this purpose, stable transplastomic tobacco plants expressing a reporter gene i.e. uidA (GUS) under four different start codons (AUG/UUG/GUG/CUG) were generated and β-glucuronidase (GUS) expression was compared. To investigate further the role of promoter sequences proximal to the start codon, uidA was expressed under two different chloroplast gene promoters psbA and psbC that use AUG and a non-AUG (GUG) start codons, respectively, and also showed significant differences in the DNA sequence surrounding the start codon. Further, to delineate the role of RNA editing that creates AUG start codon by editing non-AUG codons, if any, which is another important feature of the chloroplast transcription and translation system, transcripts were sequenced. In addition, a proteomic approach was used to identify the translation initiation site(s) of GUS and the N-terminal amino acid encoded when expressed under different non-AUG start codons. The results showed that chloroplasts use non-AUG start codons in combination with the translation initiation site as an additional layer of gene regulation to over-express proteins that are required at high levels due to their high rates of turnover.
... Accumulation of dicistronic mRNAs as well as their efficient translation leading to high levels of foreign protein has been established (Jeong et al., 2004;Staub et al., 1995). ...
... Where psbA 5' UTR has been shown to be highly effective in promoting foreign protein accumulation it was incorporated into transgene constructs as the leader sequence for a monocistronic transcription unit (Staub and Maliga, 1994;Staub and Maliga, 1995). ...
While genetic improvement of susceptible crop species may enhance resistance to microbial pathogens and facilitate reduced pesticide load, the possibility for transmission of novel genes to wild relatives has hampered acceptance of GM crops in some markets. Chloroplast transformation presents an attractive alternative to nuclear transformation and offers the potential to ameliorate these environmental concerns. Most agronomically important species exhibit maternal inheritance of organellar genomes which eliminates the threat of transgene escape through pollen. Gene silencing is absent due to site directed, single copy insertion by homologous recombination. Foreign proteins can accumulate to high levels (up to 50% of total soluble protein) and are retained within the chloroplast envelope protecting them from degradation by host cytoplasmic proteases. A bacterial chloroperoxidase gene (cpo-p) was transformed into the tobacco chloroplast genome to test its efficacy against plant pathogens and the mycotoxin producing saprophyte Aspergillus flavus.
... Initially, it was believed that the processing of such mixed precursor RNAs into monocistronic forms would determine the eventual translation of an open reading frame. However, polysome analyses and ribosome profiling experiments have demonstrated that the processing state of an mRNA has little impact on the translation rate in most cases (1,34,35,56,57). On the other hand, tRNA-K(UUU) abundance is completely independent of processing state and adjacent ORFs, enabling regulation of ribosomal protein genes across highly diverse contexts and transcripts . ...
The protein levels of chloroplast photosynthetic genes and genes related to the chloroplast genetic apparatus vary to adapt to different conditions. However, the underlying mechanisms governing these variations remain unclear. The chloroplast intron Maturase K is encoded within the trnK intron and has been suggested to be required for splicing several group IIA introns, including the trnK intron. In this study, we employed RNA immunoprecipitation followed by high-throughput sequencing (RIP-Seq) to identify MatK's preference for binding to group IIA intron domains I and VI within target transcripts. Importantly, these domains are crucial for branch point selection, and we discovered alternative branch points in three MatK target introns, the first observed instances of alternative splicing in chloroplasts. The alternative trnK lariat structure showed increased accumulation during heat acclimation. The cognate codon of tRNA-K(UUU) is highly enriched in mRNAs encoding ribosomal proteins and ribosome profiling in a trnK-matK over-expressor exhibited elevated levels of the spliced tRNA-K(UUU). Our analysis revealed a significant up-shift in the translation of ribosomal proteins compared to photosynthetic genes. Our findings suggest the existence of a novel regulatory mechanism linked to the abundance of tRNA-K(UUU), enabling the differential expression of functional chloroplast gene groups.
... Plastid transformation has various advantages over nuclear transformation (Staub and Maliga, 1995;Daniell et al., 1998;Scott and Wilkinson, 1999). At the point when, ...
For many reasons tobacco is extensively used as a model plant in transformation research and used for this study as well. Tobacco plants are cultivated in several countries on large scale. Tobacco has numerous essential traditional and modern uses in clinical field. The principle aim of this present study was to optimize the conditions for seed germination, best shooting regeneration media, optimization of antibiotics spectinomycin and kanamycin for wild type plants and in addition, to develop a protocol for efficient genetic transformation of tobacco plant cv. Petit Havana with Agrobacterium tumefaciens. Seeds of tobacco were germinated on different media without any plant growth regulators. 100% seeds germination efficiency was observed on ½ MS and best sterilization time of tobacco with ethanol was for 1 minute which also showed 100% germination efficiency. RMOP (NAA and BAP) media showed the highest shoots regeneration efficiency for both explants nodes and leaves which is nodes showed 75% and leaves showed 55% regeneration efficiency among the different media which is used for regeneration. 150 mg/L concentration of kanamycin and 500 mg/L concentration of spectinomycin for both explants (nodes and leaves) was optimized for the selection of transgenic tissues. For transformation infection time for 5 minutes and co-cultivation time period of two days showed maximum transformation efficiency of 83.33% as compared to other methods. Histochemical GUS (β-glucuronidase) assay was used to check the GUS gene expression in the nodal explants. GUS gene primers were used which amplified a 708bp fragment which also confirmed the transformation of GUS gene by Agrobacterium tumifaciens strain C58C1 containing vector p35SGUSINT. Taken together, the present study develops a tissue culture protocol and basis for Agrobacterium-mediated transformation of Nicotiana tabacum cv. Petit Havana using nodal explants.
Keywords: Tobacco, Spectinomycin, Kanamycin, Agrobacterium tumefaciens, GUS, Infection time, Co-cultivation time.
... The chloroplast transformation of C. reinhardtii with vector p463 allowed its cloning by homologous recombination (Dreesen et al. 2010). This transformation is efficient and has advantages over a nuclear transformation, including elevated levels of expression and the absence of gene silencing (Staub and Maliga 1995;Quesada-Vargas et al. 2005). ...
Bioremediation with genetically modified microalgae is becoming an alternative to remove metalloids and metals such as cadmium, a contaminant produced in industrial processes and found in domestic waste. Its removal is important in several countries including Mexico, where the San Luis Potosi region has elevated levels of it. We generated a construct with a synthetic gene for γ-glutamylcysteine synthetase and employed it in the chloroplast transformation of Chlamydomonas reinhardtii. In dose-response kinetics with media containing from 1 to 20 mg/L of cadmium, both the transplastomic clone and the wild-type strain grew similarly, but the former removed up to 32% more cadmium. While the growth of both decreased with higher concentrations of cadmium, the transplastomic clone removed 20 ± 9% more than the wild-type strain. Compared to the wild-type strain, in the transplastomic clone the activity of glutathione S-transferase and the intracellular glutathione increased up to 2.1 and 1.9 times, respectively, in media with 2.5 and 10 mg/mL of cadmium. While 20 mg/L of cadmium inhibited the growth of both, the transplastomic clone gradually duplicated. These results confirm the expression of the synthetic gene gshA in the transformed strain as revealed in its increased removal uptake and metabolic response.
... Plastid transformation has become a very practical tool that relies on site-specific insertion of foreign sequences by homologous recombination that eventually develops stable homoplasmic transplastomes. Chloroplast transformation has its own unique advantages over nuclear transformation, which includes high-level protein expression (Chiyoda et al. 2007; Lelivelt et al. 2005;Ruf et al. 2001), transgene stacking (Ruf et al. 2001;Staub and Maliga 1995;Wang et al. 2009), transgene containment, lack of position effect, and lack of epigenetic effects (Gottschamel et al. 2013;Maliga 2004). ...
In the present study, we focused on designing a species-specific chloroplast vector for Capsicum annuum L. and finding out its transformation efficiency compared to a heterologous vector. The plastid transformation vector (CaIA) was designed to target homologous regions trnA and trnI of IR region. A selectable marker gene aadA, whose expression is controlled by psbA promoter and terminator, was cloned between two flanking regions. A heterologous vector pRB95, which targets trnfM and trnG of LSC region along with aadA driven by rrn promoter and psbA terminator, was also used for developing plastid transformation in Capsicum. Cotyledonary explants were bombarded with stabilized biolistic parameters: 900 psi pressure and 9 cm flight distance, and optimized regeneration protocol (0.7 mg/L TDZ + 0.2 mg/L IAA) was used to obtain transplastomic lines on selection medium (300 mg/L spectinomycin). The aadA integration and homoplasmy were confirmed by obtaining 1.2 and 3.7 kb amplicons in CaIA transformants and subsequently verified by Southern blotting, whereas in pRB95 transformants, integration was confirmed by PCR with 1.45 kb and 255 bp amplicons corresponding to aadA integration and flanks, respectively. The transformation efficiencies attained with two plastid vectors were found to be 20%, i.e., 10 transplastomic lines in 50 bombarded plates, with CaIA and 2%, i.e., 1 transplastomic line in 50 bombarded plates, with heterologous pRB95, respectively.
... A number of selectable markers are employed in attaining stable plastid transformants that includes the aadA gene (encodes aminoglycoside 3′-adenylyltransferase) that confers resistance to spectinomycin (Goldschmidt-Clermont 1991). Other selectable marker genes used in plastid transformation are nptII (Carrer et al. 1993), codA (Serino and Maliga 1997), bar (Iamtham and Day 2000), EPSPS (Ye et al. 2001), gfp (Khan and Maliga 1999), and uidA (Staub and Maliga 1995). ...
... The plastid is considered to originate from a formerly free-living cyanobacterium and has a prokaryotic-like genetic system. Plastid transformation holds couples of unique advantages compared to conventional nuclear transformation (Staub and Maliga 1995;Scott and Wilkinson 1999;Bock 2015), e.g., remarkable high expression levels due to high polyploidy plastid genome, absence of epigenetic transgene silencing, precision of the transgene into plastid genome and the increased biosafety by maternal inheritance (Bock 2015). It has been demonstrated that GB was synthesized in plastids and GB accumulation in plastids could be more effective than in the cytosol for protecting transgenic plants against abiotic stresses . ...
Main conclusion
Plastid genome engineering is an effective method to generate drought-resistant potato plants accumulating glycine betaine in plastids.
Glycine betaine (GB) plays an important role under abiotic stress, and its accumulation in chloroplasts is more effective on stress tolerance than that in cytosol of transgenic plants. Here, we report that the codA gene from Arthrobacter globiformis, which encoded choline oxidase to catalyze the conversion of choline to GB, was successfully introduced into potato (Solanum tuberosum) plastid genome by plastid genetic engineering. Two independent plastid-transformed lines were isolated and confirmed as homoplasmic via Southern-blot analysis, in which the mRNA level of codA was much higher in leaves than in tubers. GB accumulated in similar levels in both leaves and tubers of codA-transplastomic potato plants (referred to as PC plants). The GB content was moderately increased in PC plants, and compartmentation of GB in plastids conferred considerably higher tolerance to drought stress compared to wild-type (WT) plants. Higher levels of relative water content and chlorophyll content under drought stress were detected in the leaves of PC plants compared to WT plants. Moreover, PC plants presented a significantly higher photosynthetic performance as well as antioxidant enzyme activities during drought stress. These results suggested that biosynthesis of GB by chloroplast engineering was an effective method to increase drought tolerance.
... The selection markers are chosen according to cellular autonomy and portability; thus some selection markers are dominant like the aadA gene, while other genes are recessives like the punctual mutation in the RNAr genes (rrnS and rrnL); the dominant markers are important for transformations of the highly polyploid plastomes because they increase the transformation frequency due to its effect at early stages of selection despite that may only integrate in the minority of the plastomes; on the another hand, recessive markers have lower efficiency in the transformation and only are efficient in random segregations if the plastids have sufficient copies of transformed genome [63,64]. However, the selection markers and genes of interest can be added under one promoter because the polycistronic RNAs are efficiently translated in plastids [65]; also, it is possible to obtain a transductional fusion between a resistance gene and reporter gene like gfp gene avoid phenotypic marking in transformed cells with more results in the selection process from transformed tissue [66]. ...
... Termination of rbcL transcription was originally inferred from the transformation of the plastid genome of Nicotiana tabacum [100][101][102]. When plastid vectors were targeted to the rbcL-accD intergenic region of the tobacco plastid genome [103], rbcL read-through transcripts were detectable [101][102][103][104]. However, when the insertion site was moved 170 nucleotides further downstream of rbcL, the rbcL read-through was eliminated [103]. ...
Transcription termination by the RNA polymerase (RNAP) is a fundamental step of gene expression that involves the release of the nascent transcript and dissociation of the RNAP from the DNA template. However, the functional importance of termination extends beyond the mere definition of the gene borders. Chloroplasts originate from cyanobacteria and possess their own gene expression system. Plastids have a unique hybrid transcription system consisting of two different types of RNAPs of dissimilar phylogenetic origin together with several additional nuclear encoded components. Although the basic components involved in chloroplast transcription have been identified, little attention has been paid to the chloroplast transcription termination. Recent identification and functional characterization of novel factors in regulating transcription termination in Arabidopsis chloroplasts via genetic and biochemical approaches have provided insights into the mechanisms and significance of transcription termination in chloroplast gene expression. This review provides an overview of the current knowledge of the transcription termination in chloroplasts.
... A number of selectable markers are employed in attaining stable plastid transformants that includes the aadA gene (encodes aminoglycoside 3′-adenylyltransferase) that confers resistance to spectinomycin (Goldschmidt-Clermont 1991). Other selectable marker genes used in plastid transformation are nptII (Carrer et al. 1993), codA (Serino and Maliga 1997), bar (Iamtham and Day 2000), EPSPS (Ye et al. 2001), gfp (Khan and Maliga 1999), and uidA (Staub and Maliga 1995). ...
The plastid transformation is used for high level expression of certain metabolically and industrially important recombinant proteins in plants. The vector, pFaadAII, a tobacco based vector system, harbouring a chimeric gene consisting of aadA coding region from Escherichia coli with 5′ 16S rDNA promoter and 3′ untranslated transcript region (UTR) of chlamydomonas rbcL gene, located in between the intergenic regions of rp132 and trnL genes. This vector used for transformation of plastids targets the foreign sequences to the small single-copy region of the plastome. Biolistic mode of approach for chloroplast transformation in Scoparia dulcis L., was achieved by bombarding the leaf explants and spectinomycin based selection system was used for regeneration of transformed plants. Transplastomic lines have been successfully established with overall efficiency of two transgenic lines for twenty-five bombarded explants. Integration of aadA in selection based regenerants was characterized by PCR and protein accumulation analysis along with seedlings experiment obtained from selfing. The chloroplast transformation developed in this plant system will provide scope for research in plastid based metabolic engineering pathways.
... Similar to prokaryotic gene regulation, the majority of mRNA produced in plastids is polycistronic, with proteins typically translated along the entire cistron (Staub and Maliga 1995). In one sense, the production of polycistonic mRNA is a powerful tool for metabolic engineering. ...
Owing to its small size, prokaryotic-like molecular genetics, and potential for very high transgene expression, the plastid genome (plastome) is an attractive plant synthetic biology chassis for metabolic engineering. The plastome exists as a homogenous, compact, multicopy genome within multiple-specialized differentiated plastid compartments. Because of this multiplicity, transgenes can be highly expressed. For coordinated gene expression, it is the prokaryotic molecular genetics that is an especially attractive feature. Multiple genes in a metabolic pathway can be expressed in a series of operons, which are regulated at the transcriptional and translational levels with cross talk from the plant's nuclear genome. Key features of each regulatory level are reviewed, as well as some examples of plastome-enabled metabolic engineering. We also speculate about the transformative future of plastid-based synthetic biology to enable metabolic engineering in plants as well as the problems that must be solved before routine plastome-enabled synthetic circuits can be installed.
... Likewise, unprocessed tobacco atpH and rbcL mRNAs were translated as efficiently as processed mRNAs in vitro (Yukawa et al., 2007). Furthermore, synthetic transcription units that were engineered into plastids gave rise to polycistronic transcripts that were efficiently translated in the absence of processing (e.g., Staub and Maliga, 1995). Finally, a genome-wide ribosome profiling study revealed that several polycistronic mRNAs are efficiently used as translation template, despite the known co-existence of monocistronic transcript isoforms (Zoschke and Barkan, 2015). ...
Chloroplast translation is essential for cellular viability and plant development. Its positioning at the intersection of organellar RNA and protein metabolism makes it a unique point for the regulation of gene expression in response to internal and external cues. Recently obtained high-resolution structures of plastid ribosomes, the development of approaches allowing genome-wide analyses of chloroplast translation (i.e., ribosome profiling) and the discovery of RNA-binding proteins involved in the control of translational activity have greatly increased our understanding of the chloroplast translation process and its regulation. In this review, we provide an overview of the current knowledge about the chloroplast translation machinery, its structure, organization and function. In addition, we summarize the techniques that are currently available to study chloroplast translation, describe how translational activity is controlled and which cis-elements and trans-factors are involved. Finally, we discuss how translational control contributes to the regulation of chloroplast gene expression in response to developmental, environmental and physiological cues. We also illustrate the commonalities and the differences between the chloroplast and bacterial translation machineries and the mechanisms of protein biosynthesis in these two prokaryotic systems.
... A number of selectable markers are employed in attaining stable plastid transformants that includes the aadA gene (encodes aminoglycoside 3′-adenylyltransferase) that confers resistance to spectinomycin (Goldschmidt-Clermont 1991). Other selectable marker genes used in plastid transformation are nptII (Carrer et al. 1993), codA (Serino and Maliga 1997), bar (Iamtham and Day 2000), EPSPS (Ye et al. 2001), gfp (Khan and Maliga 1999), and uidA (Staub and Maliga 1995). ...
Chloroplast transformation vectors require an expression cassette flanked by homologous plastid sequences to drive plastome recombination. The rrn16-rrn23 plastome region was selected and using this region, a new species-specific plastid transformation vector CuIA was developed with pKS+II as a backbone by inserting the rrn16-trnI and trnA-rrn23 sequences from Cucumis sativus L. An independent expression cassette with aadA gene encoding aminoglycoside 3′-adenylyltransferase with psbA controlling elements is added into the trnI-trnA intergenic region that confers resistance to spectinomycin. An efficient plastid transformation in bitter melon (Momordica charantia L.) was achieved by bombardment of petiole segments. The frequency of transplastomic plants yielded using standardized biolistic parameters with CuIA vector was two per 15 bombarded plates, each containing 20 petiole explants. Integration of aadA gene was verified by PCR analysis in transplastomes. Transplastomic technology developed may be a novel approach for high level expression of pharmaceutical traits.
... Even though the technology significantly 150 improved since 1998, no transplastomic clones were obtained until ACC2 defective leaf tissue 151 was used for bombardments (Table 1) , providing overwhelming support for the absence of 152 ACC2 activity being critical for high frequency plastid transformation in Arabidopsis thaliana. (Staub and Maliga, 1995). Thus, GFP accumulation was anticipated only ...
Plastid transformation is routine in tobacco, but 100-fold less frequent in Arabidopsis, preventing its use in plastid biology. A recent study revealed that null mutations in ACC2, encoding a plastid-targeted acetyl-CoA-carboxylase, cause hypersensitivity to spectinomycin. We hypothesized that plastid transformation efficiency should increase in the acc2 background, because when ACC2 is absent, fatty acid biosynthesis becomes dependent on translation of the plastid-encoded ACC β-Carboxylase subunit. We bombarded ACC2-defective Arabidopsis leaves with a vector carrying a selectable spectinomycin resistance (aadA) gene and gfp, encoding the green fluorescence protein GFP. Spectinomycin resistant clones were identified as green cell clusters on a spectinomycin medium. Plastid transformation was confirmed by GFP accumulation from the second open reading frame of a polycistronic mRNA, that would not be translated in the cytoplasm. We obtained one to two plastid transformation events per bombarded sample in spectinomycin hypersensitive Slavice (Sav-0) and Columbia acc2 knockout backgrounds, an approximately 100-fold enhanced plastid transformation frequency. Sav-0 and Columbia are accessions in which plant regeneration is uncharacterized or difficult to obtain. A practical system for Arabidopsis plastid transformation will be obtained by creating an ACC2 null background in a regenerable Arabidopsis accession. The recognition that the duplicated ACCase in Arabidopsis is an impediment to plastid transformation provides a rational template to implement plastid transformation in related recalcitrant crops.
... Note that not all elements depicted in Figure 2 are indispensable. Thus, separate promoters are not required, if transcription is mediated by endogenous transcription start signals (Staub and Maliga, 1995). Such "operon extension vectors" are described in detail by Herz et al. 2005). ...
January of 1983 was a turning point for plant biotechnology when tobacco was immortalized as a surrogate biological system for testing gene function at a conference on “Advances in Gene Technology: Molecular Genetics of Plants and Animals” hosted by the Miami Winter Symposia series. Although Arabidopsis has now become the system of choice for nuclear gene integration due to the ease of transformation and a short generation cycle, tobacco remains the only established system for plastid transformation. This review summarizes the use of tobacco in dissecting plant biology concepts pertaining to the three important compartments of the cell that harbor genetic material within them. Recent studies in N. benthamiana have brought the genus back to the limelight as an outstanding system for transient protein expression. Overall, this chapter also brings out the advantages and limitations of tobacco as a system for discovery in plant biology. As a nonfood and nonfeed crop tobacco retains a remarkable potential for use as a biofactory. Ironically, this genus with a notorious health reputation may prove to be indispensable for the production of medically relevant compounds.
... There is a great potential, therefore, for the genetic manipulation of key enzymes involved in stress metabolism in plants within plastids. Because plastid genomes of major crops including cotton and soybean have been Nicotiana tabacum nptII [153] Nicotiana tabacum uidA [154] Nicotiana tabacum Human somatotropin (hST) [155] Nicotiana tabacum cry [88] Nicotiana tabacum cry9Aa2 [84] Nicotiana tabacum Bar & aadA [156] Nicotiana tabacum Cor 15a-FAD7 [157] Nicotiana tabacum rbcL [55] Nicotiana tabacum DXR [158] Nicotiana tabacum aadA & gfp [60] Nicotiana tabacum Delta(9) desaturase [159] Nicotiana tabacum AsA2 [160] Nicotiana tabacum PhaG & PhaC [161] Nicotiana tabacum gfp [4] Nicotiana tabacum A1AT [162] Arabidopsis thaliana aadA [39] Solanum tuberosum aadA & gfp [40] Oryza sativa aadA & gfp [50] Solanum lycopersicon aadA [1] Solanum lycopersicon Lyc [42] Brassica napus aadA & cry1Aa10 [44] Brassica napus aadA [38] Lesquerella fendleri aadA & gfp [43] Daucus carota dehydrogenase (badh) [48] Gossypium hirsutum aphA-6 [49] Glycine max aadA [47] Petunia hybrida aadA & gusA [45] Lactuca sativa gfp [46] Brassica oleracea gus & aadA [163] Lettuce gfp [164] Populus alba gfp [51] Brassica oleracea aadA & uidA [74] Beta vulgaris aadA & uidA [165] Crocus sativus CstLcyB1& CstLcyB2a [166] Solanum melongena aadA [26] Arabidopsis thaliana pre-Tic40-His [167] Zea mays ManA [168] successfully transformed, this offers an exciting new approach to create transgenic plants with abiotic stress tolerance [93]. Therefore, the authors believe that there appears to be tremendous potential for increasing tolerance in plants to a number of stresses by expression of appropriate genes within plastids due to the maternal inheritance of transgenes that confer tolerance to abiotic stress. ...
... In contrast, in the pZB plants where the Prrn promoter sequence is shorter (132-bp long), continued transcription through the rbcL-3¢-UTR was observed producing approximately equal amounts of mono-(1.4 kb) and tri-(3.1 kb) cistronic message (Fig. 3). It is worth noting that similar to our observation with the Chlamydomonas rbcL 3¢-UTR, the corresponding 3¢-UTR from tobacco rbcL has also been shown to display inefficient termination, resulting in polycistronic mRNAs (Staub and Maliga 1995). ...
;The small subunit (SS) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a nuclear gene-encoded protein that is imported into chloroplasts where it assembles with the large subunit (LS) after removal of the transit peptide to form Rubisco. We have explored the possibility that the severe deficiency in photosynthesis exhibited in nuclear transgenic tobacco (line 5) expressing antisense rbcS coding DNA that results in low SS and Rubisco protein content [Rodermel et al. (1988) Cell 55: 673] could be complemented by introducing a copy of the rbcS gene into its plastid genome through chloroplast transformation. Two independent lines of transplastomic plants were generated, in which the tobacco rbcS coding sequence, either with or without the transit sequence, was site-specifically integrated into the plastid genome. We found that compared with the antisense plants, expression of the plastid rbcS gene in the transplastomic plants resulted in very high mRNA abundance but no increased accumulation of the SS and Rubisco protein or improvement in plant growth and photosynthesis. Therefore, there is a limitation in efficient translation of the rbcS mRNA in the plastid or an incorrect processing and modification of the plastid-synthesized SS protein that might cause its rapid degradation.
... While site-directed integration through homologous recombination appears to be a requirement, it might also provide more control of genetic engineering and increased uniformity of transgene expression (8,75,78,79). Additionally, one can make use of polycistronic operons, much like a bacterial system, to permit coordinated expression of multiple genes (79,80). ...
A major focus of biotechnology is the improvement of human health around the globe. It is anticipated that the genomic revolution will greatly expand our knowledge of the molecular basis of many diseases and pathological states. Combining this knowledge with powerful screening techniques will be used in the development of safe and efficacious biologics and drugs for the prevention and treatment of disease. Unfortunately, the availability of these new biologics and drugs for use by all those who need them greatly depends on economic considerations such as the cost of their development, production, and delivery. Therefore, a major challenge of biotechnology is to translate clinical innovations to economically viable practice. The production of plant-derived vaccines for mucosal delivery is a step toward that goal.
... On the other hand, polycistronic translation is common in prokaryotes, since ribosomes enter the RNA internally at a "Shine-Dalgarno Sequence" and several of these sequences, each preceding a coding region, can be accommodated per RNA molecule. Since chloroplasts are of prokaryotic origin, they can use the prokaryotic translation mechanism (Staub et al., 1995). Thus they could be used for expressing polycistronic RNA as soon as chloroplast transformation technology is available. ...
... By carefully choosing the insertion site in the plastid genome, this approach can result in high levels of mRNA and can give extremely high yields of protein expressed from the transgene. An early description of this type of system demonstrated that a promoterless uidA gene inserted downstream of the plastid rbcL gene resulted in approximately 4-fold higher β-glucuronidase protein levels than a construct containing a heterologous ribosomal promoter inserted at the same site in the plastid genome, despite a greatly increased concentration of monocistronic uidA mRNA in the latter case (Staub and Maliga, 1995). A number of researchers have since used promoterless constructs taking advantage of read-through transcription from the native plastid ribosomal or psbA promoters to achieve high-level protein accumulation in plastid transformants, demonstrating the utility of this approach (Herz et al., 2005;Chakrabarti et al., 2006;Gray et al., 2009Gray et al., , 2011. ...
Many efforts are underway to engineer improvements in photosynthesis to meet the challenges of increasing demands for food
and fuel in rapidly changing environmental conditions. Various transgenes have been introduced into either the nuclear or
plastid genomes in attempts to increase photosynthetic efficiency. We examine the current knowledge of the critical features
that affect levels of expression of plastid transgenes and protein accumulation in transplastomic plants, such as promoters,
5’ and 3’ untranslated regions, RNA-processing sites, translation signals and amino acid sequences that affect protein turnover.
We review the prior attempts to manipulate the properties of ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) through
plastid transformation. We illustrate how plastid operons could be created for expression of the multiple genes needed to
introduce new pathways or enzymes to enhance photosynthetic rates or reduce photorespiration. We describe here the past accomplishments
and future prospects for manipulating plant enzymes and pathways to enhance carbon assimilation through plastid transformation.
... The endonucleolytic and intercistronic cleavage of the psbA 5'UTR in vivo first reported here is an important step for mRNA stability and translation. Most chloroplast genes from higher plants are organized in clusters and are primarily co-transcribed as polycistronic forms and endonucleolytica[ly processed into monocistronic mRNAs, during which some of the transcripts are edited and/or spliced (Barkan, 1988; Ruf et al., 1994)Staub and Maliga, 1995). Processing of the psbA 5'UTR occurs just upstream of a consensus Shine-Da[gano sequence in Chlamydomonas (Bruick and Mayfield, 1998). ...
Post-transcriptional RNA processing and translational regulations are important steps for gene expression. To analyze the
5′UTRof psbA that enhances translation of the sweet protein monellin in chloroplasts, we cloned the monellin gene, with and without
thepsbA 5′UTR, into the chloroplast expression vector for chloroplast transformation. Transgenic plants were identified as being
transplastomic via PCR and Southern blot analyses. We also observed non-specific recombination during tobacco chloroplast
transformation. Analyses of the transcription patterns showed that intercistronic cleavage of the psbA mRNA 5′ untranslated
(UTR) region was functional at the mature stage, with the monocistronic mRNA ofmonellin increasing while its dicistronic mRNA decreased. Moreover, monellin accumulation accounted for 2.3% of the total soluble
protein at the mature stage, but only 1.3% at the young stage in transplastomic lines that contained the 5′UTRof psbA. These results suggest that activation of the endonucleolytic cleavage of thepsbA 5′UTR element depends on chloroplast developmental conditions, and that it enhances the accumulation of sweet protein monellin
in those chloroplasts.
... The accumulation of both peptides in pCWEA1-transformed plants remains to be demonstrated. However, in carrot protoplasts (Putterill & Gardner 1989) and tobacco plastids (Staub & Maliga 1995) such an internal open reading frame can be efficiently translated. The antimicrobial actions of ESF12 and Ac-AMP1 are different because Ac-AMP1 is a chitin-binding, defensin-like peptide (Broekaert et al. 1992), while the predicted structure of ESF12 contains an amphipathic α-helix thought to form channels in biological membranes (Powell et al. 1995). ...
Plasmids, pCA1 and pCWEA1, carrying antimicrobial peptide gene(s), Ac-AMP1.2 and ESF12, were used to transform hybrid poplar clones Ogy and NM6. Peptide Ac-AMP1.2 is an analog of Ac-AMP1 which is one of the smallest chitin-binding proteins. Synthetic peptide ESF12 mimics the amphipathic -helix found in magainins. Transgene mRNA was detected in the transformed plants. When evaluated for resistance to hybrid poplar pathogen Septoria musiva with an invitro leaf disk assay, the transformed Ogy plants showed significantly increased pathogen resistance as compared to the untransformed Ogy.
... This leads to the possibility that multiple gene expression involving polycistronic transgenes may be feasible in chloroplasts. This possibility has been tested by Staub and Maliga (1995). These authors reported the efficient expression of a promoter-distal GUS cistron that was part of a chimeric rbcL-uidA gene that had been introduced into the tobacco chloroplast genome. ...
With an ever-increasing frequency, it has become desirable to express many foreign genes in the same transgenic plant. A range
of approaches to this problem have been eplored in the period since gene transfer was developed as a useful tool in plant
science. These have ranged from assembly of multiple transcription units on a single DNA vector, to the execution of several
independent transformations involving single genes followed by successive rounds of hybridization to yield plant lines with
the desired combination of genes, to cotransformation with multiple plasmids followed by molecular screening to identify desired
transgenic plants. Recent developments have provided new alternatives to the problem of the expression of multiple foreign
genes in plants, approaches that revolve around the expression of polycistronic mRNAs. This review summarizes the current
status of the area of polycistronic gene expression in transgenic plants, within the context of multigene expression.
... Nicotiana tabacum nptII [153] Nicotiana tabacum uidA [154] Nicotiana tabacum Human somatotropin (hST) [155] Nicotiana tabacum cry [88] Nicotiana tabacum cry9Aa2 [84] Nicotiana tabacum Bar & aadA [156] Nicotiana tabacum Cor 15a-FAD7 [157] Nicotiana tabacum rbcL [55] Nicotiana tabacum DXR [158] Nicotiana tabacum aadA & gfp [60] Nicotiana tabacum Delta(9) desaturase [159] Nicotiana tabacum AsA2 [160] Nicotiana tabacum PhaG & PhaC [161] Nicotiana tabacum gfp [4] Nicotiana tabacum A1AT [162] Arabidopsis thaliana aadA [39] Solanum tuberosum aadA & gfp [40] Oryza sativa aadA & gfp [50] Solanum lycopersicon aadA [1] Solanum lycopersicon Lyc [42] Brassica napus aadA & cry1Aa10 [44] Brassica napus aadA [38] Lesquerella fendleri aadA & gfp [43] Daucus carota dehydrogenase (badh) [48] Gossypium hirsutum aphA-6 [49] Glycine max aadA [47] Petunia hybrida aadA & gusA [45] Lactuca sativa gfp [46] Brassica oleracea gus & aadA [163] Lettuce gfp [164] Populus alba gfp [51] Brassica oleracea aadA & uidA [74] Beta vulgaris aadA & uidA [165] Crocus sativus CstLcyB1& CstLcyB2a [166] Solanum melongena aadA [26] Arabidopsis thaliana pre-Tic40-His [167] Zea mays ManA [168] successfully transformed, this offers an exciting new approach to create transgenic plants with abiotic stress tolerance [93]. Therefore, the authors believe that there appears to be tremendous potential for increasing tolerance in plants to a number of stresses by expression of appropriate genes within plastids due to the maternal inheritance of transgenes that confer tolerance to abiotic stress. ...
Genetic material in plants is distributed into nucleus, plastids and mitochondria. Plastid has a central role of carrying out photosynthesis in plant cells. Plastid transformation is becoming more popular and an alternative to nuclear gene transformation because of various advantages like high protein levels, the feasibility of expressing multiple proteins from polycistronic mRNAs, and gene containment through the lack of pollen transmission. Recently, much progress in plastid engineering has been made. In addition to model plant tobacco, many transplastomic crop plants have been generated which possess higher resistance to biotic and abiotic stresses and molecular pharming. In this mini review, we will discuss the features of the plastid DNA and advantages of plastid transformation. We will also present some examples of transplastomic plants developed so far through plastid engineering, and the various applications of plastid transformation.
Expression of transgenes from the plastid genome offers a number of attractions to biotechnologists, with the potential to attain very high protein accumulation levels arguably being the most attractive one. High-level transgene expression is of particular importance in resistance engineering (e.g., for expression of insecticidal proteins) and molecular farming (e.g., for expression of pharmaceutical proteins and industrial enzymes). Over the past decades, the production of many commercially valuable proteins in chloroplast-transgenic (transplastomic) plants has been attempted, including pharmaceutical proteins (e.g., subunit vaccines and protein antibiotics) and industrial enzymes. Although in some cases, spectacularly high foreign protein accumulation levels have been obtained, expression levels were disappointingly poor in other cases. In this review, I summarize our current knowledge about the factors influencing the efficiency of plastid transgene expression, and highlight possible optimization strategies to alleviate problems with poor expression levels. I also discuss available techniques for inducible expression of chloroplast transgenes.
Today global agriculture is facing downfall in total production and one of the critical factors for less productivity is food crop damages caused by insects and pests. Food availability is one of the major objectives to make food security attainable to people of the world and thereby huge money is spent every year to control the population of crop-damaging insects. Despite all the insect–pest control measures, there are considerable losses in agricultural crops every year. A chunk of money is spent worldwide on the use of agrochemicals which not only pollute the environment but also pose severe threats to human and animal health. Additionally, their injudicious and indiscriminate uses have also become responsible for resurgence and resistance in insects in many corners of the world. Therefore, it becomes necessary to control the population densities of crop-damaging insects by employing more programmed, eco-friendly, and effective crop protection strategies.
Plant genetic transformation is an important technological advancement in modern science, which has not only facilitated gaining fundamental insights into plant biology but also started a new era in crop improvement and commercial farming. However, for many crop plants, efficient transformation and regeneration still remain a challenge even after more than 30 years of technical developments in this field. Recently, FokI endonuclease-based genome editing applications in plants offered an exciting avenue for augmenting crop productivity but it is mainly dependent on efficient genetic transformation and regeneration, which is a major roadblock for implementing genome editing technology in plants. In this chapter, we have outlined the major historical developments in plant genetic transformation for developing biotech crops. Overall, this field needs innovations in plant tissue culture methods for simplification of operational steps for enhancing the transformation efficiency. Similarly, discovering genes controlling developmental reprogramming and homologous recombination need considerable attention, followed by understanding their role in enhancing genetic transformation efficiency in plants. Further, there is an urgent need for exploring new and low-cost universal delivery systems for DNA/RNA and protein into plants. The advancements in synthetic biology, novel vector systems for precision genome editing and gene integration could potentially bring revolution in crop-genetic potential enhancement for a sustainable future. Therefore, efficient plant transformation system standardization across species holds the key for translating advances in plant molecular biology to crop improvement.
The plastid genome of higher plants is a 120-kb to 160-kb double-stranded DNA present in 1,900 to 50,000 copies per leaf cell. To obtain genetically stable transplastomic lines every one of the plastid genome copies (ptDNA) should be uniformly altered in a plant. Transformation is accomplished through the following steps: (i) introduction of the transforming DNA, encoding antibiotic resistance, by the biolistic process or PEG treatment; (ii) integration of the transforming DNA by two homologous recombination events and (iii) elimination of wild-type genome copies during repeated cell divisions on a selective medium. As integration of foreign DNA always occurs by homologous recombination, plastid transformation vectors contain segments of the plastid genome to target insertions to specific locations. The plastid vectors also contain a marker for selection. Useful, non-selectable genes are cloned next to selectable marker genes, with which they are introduced into the plastid genome. Homology-directed genome manipulations have included introduction of point-mutations and deletion of targeted genes.
AGL6 is an ancient subfamily of MADS-box genes found in both gymnosperms and angiosperms. Its functions remained elusive despite
the fact that the MADS-box genes and the ABC model have been studied for >20 years. Nevertheless, recent discoveries in petunia,
rice, and maize support its involvement in the ‘E’ function of floral development, very similar to the closely related AGL2 (SEPALLATA) subfamily which has been well characterized. The known functions of AGL6 span from ancient conserved roles to new functions acquired in specific plant families. The AGL6 genes are involved in floral meristem regulation, in floral organs, and ovule (integument) and seed development, and have
possible roles in both male and female germline and gametophyte development. In grasses, they are also important for the development
of the first whorl of the flower, whereas in Arabidopsis they may play additional roles before floral meristem formation.
This review covers these recent insights and some other aspects that are not yet fully elucidated, which deserve more studies
in the future.
When Erwin Baur, at the beginning of this century, proposed that the non-Mendelian inheritance of leaf variegations can be explained with the assumption that chloroplasts (plastids) contain their own genetic information (Baur 1909, 1910), he found himself confronted with the sheer disbelief of many of his colleagues (Hagemann 1999). It took more than half a century until the discovery of chloroplast DNA (Chun et al. 1963; Sager and Ishida 1963) provided the ultimate proof for Baur’s ingenious hypothesis. Already with the very first analyses on chloroplast DNA sequences, it became obvious that plastid and eubacterial genomes are evolutionarily related (Schwarz and Kössel 1979, 1980), a finding that provided direct molecular evidence for the endosymbiotic origin of organelles (Gray 1989). The elucidation of the complete DNA sequence of two chloroplast genomes in 1986 (Ohyama et al. 1986; Shinozaki et al. 1986) marks a milestone in organelle genetics and has had a profound influence on our understanding of the biology and evolution of plastids (cf. Hagemann and Hagemann 1994; Hagemann et al. 1996, 1998).
We developed a new plastid transformation vector system using the putative replication origin of a minicircular chromosome from the marine dinoflagellate Heterocapsa
triquetra. Transplastomic tobacco plants generated with this vector properly expressed the green fluorescent protein (GFP) gene without incorporating it into the plastid genome. To construct the episomal vector, a 610-bp DNA fragment containing the putative replication origin was fused to a dicistronic expression cassette encoding the aminoglycoside 3′-adenyltransferase (aadA) and gfp genes under control of the plastid rrn promoter. The vector was delivered to plastids of tobacco leaf explants by biolistic bombardment. After 8 weeks of bombardment, episomal transformant shoots were generated from leaf explants cultured on selection media containing 500 mg/L spectinomycin. Fluorescence microscopy and northern blot analysis demonstrated GFP expression in episomal transformant plants. PCR, Southern blot analysis, recovery of episomes, and sequencing analysis showed the vector to be maintained as self-replicating extrachromosomal circular DNA molecules for at least 6 months. Using a single construct for all plants, our episomal vector system may offer an advantage over the conventional plastid vector systems, which require species-specific constructs.
Approximately ten years have elapsed since the initiation of the first experiments which led to the production of fertile transgenic maize plants. The technology is now in widespread use for both commercial and academic purposes. Here, some of the key events leading to the production of fertile transgenic maize are reviewed, as well as significant improvements which have followed in subsequent years. Finally, areas in which additional breakthroughs are needed and likely to be developed within the next decade are discussed.
Plant research has profited enormously from the options provided by genetic engineering methods. Overexpression of genes from the nucleus or their targeted knock-down by RNA interference has enabled the dissection of individual functions of genes or networks, boosting our understanding of plant genetics and physiology. However, a fraction of the plants genome is still encoded in their organelles, namely mitochondria and plastids. Genetic engineering of the plastid genome became feasible in 1990 and since then it has enabled numerous studies on plastid-encoded gene functions and also on biotechnological approaches for recombinant protein production. Although, plastid transformation differs largely from nuclear transformation regarding vector requirements, DNA delivery, and host range. Our intention is to give an overview about the techniques applied, the options, and the drawbacks of plastid transformation.
Food production has to be significantly increased in order to feed the fast growing global population estimated to be 9.1 billion by 2050. The Green Revolution and the development of advanced plant breeding tools have led to a significant increase in agricultural production since the 1960s. However, hundreds of millions of humans are still undernourished, while the area of total arable land is close to its maximum utilization and may even decrease due to climate change, urbanization, and pollution. All these issues necessitate a second Green Revolution, in which biotechnological engineering of economically and nutritionally important traits should be critically and carefully considered. Since the early 1990s, possible applications of plastid transformation in higher plants have been constantly developed. These represent viable alternatives to existing nuclear transgenic technologies, especially due to the better transgene containment of transplastomic plants. Here, we present an overview of plastid engineering techniques and their applications to improve crop quality and productivity under adverse growth conditions. These applications include (1) transplastomic plants producing insecticidal, antibacterial, and antifungal compounds. These plants are therefore resistant to pests and require less pesticides. (2) Transplastomic plants resistant to cold, drought, salt, chemical, and oxidative stress. Some pollution tolerant plants could even be used for phytoremediation. (3) Transplastomic plants having higher productivity as a result of improved photosynthesis. (4) Transplastomic plants with enhanced mineral, micronutrient, and macronutrient contents. We also evaluate field trials, biosafety issues, and public concerns on transplastomic plants. Nevertheless, the transplastomic technology is still unavailable for most staple crops, including cereals. Transplastomic plants have not been commercialized so far, but if this crop limitation were overcome, they could contribute to sustainable development in agriculture
Expression of transgenes from the plastid genome offers a number of attractions to biotechnologists, with the potential to attain very high protein accumulation levels arguably being the most attractive one. High-level transgene expression is of particular importance in resistance engineering (e.g., via expression of insecticidal proteins) and molecular farming. Over the past years, the production of many commercially valuable proteins in chloroplast-transgenic (transplastomic) plants has been attempted, including pharmaceutical proteins (such as subunit vaccines and protein antibiotics) and industrial enzymes. Although, in some cases, spectacularly high foreign protein accumulation levels have been obtained, expression levels were disappointingly poor in other cases. In this review, I summarize our current knowledge about the factors influencing the efficiency of plastid transgene expression and highlight possible optimization strategies to alleviate problems with poor expression levels.
Overall translational machinery in plastids is similar to that of E. coli. Initiation is the crucial step for translation and this step in plastids is somewhat different from that of E. coli. Unlike the Shine-Dalgarno sequence in E. coli, cis-elements for translation initiation are not well conserved in plastid mRNAs. Specific trans-acting factors are generally required for translation initiation and its regulation in plastids. During translation elongation, ribosomes pause sometimes on photosynthesis-related mRNAs due probably to proper insertion of nascent polypeptides into membrane complexes. Codon usage of plastid mRNAs is different from that of E. coli and mammalian cells. Plastid mRNAs do not have the so-called rare codons. Translation efficiencies of several synonymous codons are not always correlated with codon usage in plastid mRNAs.
We describe here the development of a reproducible plastid transformation system for potato and regeneration of plants with uniformly transformed plastids. Two distinct tobacco-specific plastid vectors, pZS197 (Prrn/aadA/TpsbA) and pMON30125 (Prrn/GFP/ Trps16::PpsbA/aadA/TpsbA), designed for integration into the large single copy and inverted repeat regions of the plastid genome, respectively, were bombarded into leaf explants of potato line FL1607. A total of three transgenic lines were selected out of 46 plates bombarded with pZS197 and three transgenic lines out of 104 plates were obtained with pMON30125. Development of a high frequency leaf-based regeneration system, a stringent selection scheme and optimization of biolistic transformation protocol were critical for recovery of plastid transformants. Plastidexpressed green fluorescent protein was used as a visual marker for identification of plastid transformants at the early stage of selection and shoot regeneration. The establishment of a plastid transformation system in potato, which has several advantages over routinely used nuclear transformation, offers new possibilities for genetic improvement of this crop.
The engineering of metabolic pathways in plants often requires the concerted expression of more than one gene. While with traditional transgenic approaches, the expression of multiple transgenes has been challenging, recent progress has greatly expanded our repertoire of powerful techniques making this possible. New technological options include large-scale co-transformation of the nuclear genome, also referred to as combinatorial transformation, and transformation of the chloroplast genome with synthetic operon constructs. This review describes the state of the art in multigene genetic engineering of plants. It focuses on the methods currently available for the introduction of multiple transgenes into plants and the molecular mechanisms underlying successful transgene expression. Selected examples of metabolic pathway engineering are used to illustrate the attractions and limitations of each method and to highlight key factors that influence the experimenter's choice of the best strategy for multigene engineering.
Vitamin E (tocopherol: Toc) is an important lipid-soluble antioxidant synthesized in chloroplasts. Among the 8 isoforms of vitamin E, α-Toc has the highest activity in humans. To generate transgenic plants with enhanced vitamin E activity, we applied a chloroplast transformation technique. Three types of the transplastomic tobacco plants (pTTC, pTTMT and pTTC-TMT) carrying the Toc cyclase (TC) or γ-Toc methyltransferase (γ-TMT) gene and the TC plus γ-TMT genes as an operon in the plastid genome, respectively, were generated. There was a significant increase in total levels of Toc due to an increase in γ-Toc in the pTTC plants. Compared to the wild-type plants, Toc composition was altered in the pTTMT plants. In the pTTC-TMT plants, total Toc levels increased and α-Toc was a major Toc isoform. Furthermore, to use chloroplast transformation to produce α-Toc-rich vegetable, TC-overexpressing transplastomic lettuce plants (pLTC) were generated. Total Toc levels and vitamin E activity increased in the pLTC plants compared with the wild-type lettuce plants. These findings indicated that chloroplast genetic engineering is useful to improve vitamin E quality and quantity in plants.
Soybean is the most valuable grain legume crop, representing the primary source of protein and vegetable oil throughout the world. In the past decades, soybean crops have been improved significantly in agronomic traits such as yield, disease and pest resistance, and seed composition by both conventional and transgenic breeding methods. In this chapter, we describe the history of soybean development and distribution, trait improvement by conventional breeding, the advancement of transformation and transgenic traits, current and future soybean products, and new technologies in modern agricultural practices. New transgenic soybean products with stacking traits can further benefit human life, environment preservation, and global economic advancement. Once biotechnology has gained widespread acceptance, the miracle soybean will fully express its beauty.
A major challenge of biotechnology is to reduce clinical innovations to economically viable practice. While still in experimental stages of development, edible vaccines stand as an excellent example of this maxim: merging innovations in medical science and plant biology, to create efficacious and affordable pharmaceuticals. In this chapter, we present an updated report on advances in this field and in particular on the recent clinical trials. We will then outline areas of research that deserve further attention.
Biolistic delivery of DNA initiated plastid transformation research and still is the most widely
used approach to generate transplastomic lines in both algae and higher plants. The principal design
of transformation vectors is similar in both phylogenetic groups. Although important additions to
the list of species transformed in their plastomes have been made in algae and in higher plants, the
key organisms in the area are still the two species, in which stable plastid transformation was initially
successful, i.e., Chlamydomonas reinhardtii and tobacco. Basic
research into organelle biology has substantially benefited from the homologous recombination-based
capability to precisely insert at predetermined loci, delete, disrupt, or exchange plastid genome
sequences. Successful expression of recombinant proteins, including pharmaceutical proteins, has
been demonstrated in Chlamydomonas as well as in higher plants,
where some interesting agronomic traits were also engineered through plastid transformation.
Plastids are surrounded by an envelope consisting of a double membrane. This barrier has to be penetrated or overcome by the
DNA when transforming the plastome. Both the biolistic and polyethylene glycol-mediated transformation techniques accomplish
this task, albeit by different mechanisms. We were the first laboratory to successfully use the polyethylene glycol (PEG)-method
for plastid transformation, yet we use the particle gun when appropriate. In this report we compare the two methods and discuss
their shortcomings and advantages. Plastid transformations with various constructs, mainly using theaadA gene as a selective marker, were performed. We point to potential problems likely to be encountered during the transformation
and selection processes and offer possibilities for improvement. We give further examples of the successful application of
plastome transformation and show its merits in addressing biological questions concerning the elucidation of plastid sequences
of unknown function and the control of plastid gene expression.
Experiments on fusion of mesophyllic protoplasts of Solanum tuberosum (Lugovskoi and Slavyanka cultivars) possessing the nptII gene in the nuclear DNA with transplastome Solanum rickii plants (which possess the aadA gene) that we have derived previously, are performed. Hybrid plants with the genes aadA and nptII, the chloroplasts of S. rickii and S. tuberosum, and a hybrid nuclear genome were obtained in a selection medium containing the antibiotics kanamycin, spectomycin, and streptomycin.
The result is confirmed by results of PCR analyses.
The complex genomes of many economically important crops present tremendous challenges to understand the genetic control of many quantitative traits with great importance in crop production, adaptation, and evolution. Advances in genomic technology need to be integrated with strategic genetic design and novel perspectives to break new ground. Complementary to individual-gene-targeted research, which remains challenging, a global assessment of the genomic distribution of trait-associated SNPs (TASs) discovered from genome scans of quantitative traits can provide insights into the genetic architecture and contribute to the design of future studies. Here we report the first systematic tabulation of the relative contribution of different genomic regions to quantitative trait variation in maize. We found that TASs were enriched in the nongenic regions, particularly within a 5-kb window upstream of genes, which highlights the importance of polymorphisms regulating gene expression in shaping the natural variation. Consistent with these findings, TASs collectively explained 44%-59% of the total phenotypic variation across maize quantitative traits, and on average, 79% of the explained variation could be attributed to TASs located in genes or within 5 kb upstream of genes, which together comprise only 13% of the genome. Our findings suggest that efficient, cost-effective genome-wide association studies (GWAS) in species with complex genomes can focus on genic and promoter regions.
Plant genetic engineering has contributed substantially to the understanding of gene regulation and plant development, in
the generation of transgenic organisms for widespread usage in agriculture, and has increased the potential uses of crops
for industrial and pharmaceutical purposes. As the application of geneticallly engineered plants has widened, so has the need
to develop methods to fine-tune control of transgene expression. The availability of a broad spectrum of promoters that differ
in their ability to regulate the temporal and spatial expression patterns of the transgene can dramatically increase the successful
application of transgenic technology. Indeed, a variety of promoters in necessary at all levels of genetic engineering in
plants, from basic research discoveries, concepts and question to development of economically viable crops and plant commodities,
to addressing legitimate concerns raised about the safety and containment of transgenic plants in the environment. This review
covers the characterization and usage of a broad range of promoters employed in plant genetic engineering, including the widespread
use of plant promoters with viral and plant origin that drive constitutive expression. Also covered are selected tissue-specific
promoters from fruit, seed and grain, tubers, flowers, pistils, anther and pollen, roots and root nodules, and leaves and
green tissue. Topics also include organellar promoters, and those found in specific cell types, as well as the development
and evaluation of inducible (endogenous and exogenous origin) and synthetic plant promoter systems. Discussions on the relevance
and potential pitfalls within specific applications are included.
Understanding how the information contained in genes is mapped onto the phenotypes, and deriving formal frameworks to search
for generic aspects of developmental constraints and evolution remains one of the main challenges of contemporary biological
research. The Mexican endemic triurid Lacandonia schismatica (Lacandoniaceae), a mycoheterotrophic monocotyledonous plant with hermaphroditic reproductive axes is alone among 250 000
species of angiosperms, as it has central stamens surrounded by a peripheral gynoecium, representing a natural instance of
a homeotic mutant. Based on the classical ABC model of flower development, it has recently been shown that the B-function
gene APETALA3 (AP3), essential for stamen identity, was displaced toward the flower centre in L. schismatica (ABC to ACB) from the early stages of flower development. A functional conservation of B-function genes from L. schismatica through the rescue of B-gene mutants in Arabidopsis thaliana, as well as conserved protein interactions, has also been demonstrated. Thus, it has been shown that relatively simple genetic
alterations may underlie large morphological shifts fixed in extant natural populations. Nevertheless, critical questions
remain in order to have a full and sufficient explanation of the molecular genetic mechanisms underlying L. schismatica’s unique floral arrangement. Evolutionary approaches to developmental mechanisms and systems biology, including high-throughput
functional genomic studies and models of complex developmental gene regulatory networks, constitute two main approaches to
meet such a challenge. In this review, the aim is to address some of the pending questions with the ultimate goal of investigating
further the mechanisms of L. schismatica’s unique homeotic flower arrangement and its evolution.
Summary The gene for cytochromeb-559, associated with the photosystem II reaction center, has been located on the spinach plastid chromosome by cell-free
coupled transcription-translation and RNA-programmed hybrid selection translation using appropriate recombinant DNAs, RNA
fractions, and monospecific antisera. The gene is located in the large single-copy segment of the plastid chromosome between
the genes for cytochomef and the P680 chlorophylla apoprotein of photosystem II and transcribed in the opposite direction relative to these genes. The 10 kd protein is decoded
from a bicistronic 1.0 kb mRNA and is apparently not made as a precursor in cell-free rabbit reticulocyte andE. coli lysates.
The N-terminal amino acid sequence of a 3.2 kDa photosystem II polypeptide is shown to be identical to that of a polypeptide encoded by an open reading frame of 38 codons (orf38) in wheat chloroplast DNA. Orf38 is located just downstream of the psbE and psbF genes for the polypeptides of cytochrome b-559. Analysis of the transcription of this region of chloroplast DNA shows that psbE, psbF and orf38 are co-transcribed to give a 1.1 kb polycistronic transcript which also contains another open reading frame of 40 codons. The orf38 and orf40 products are hydrophobic polypeptides which are both predicted to span the thylakoid membrane once. Orf38 and orf40 are highly conserved, and map to similar locations adjacent to psbE and psbF, in all organisms from which this region of DNA has been sequenced. We propose that orf38 is named psbL.
The plastid genome of higher plants is relatively small, 120–230-kb in size, and present in up to 10,000 copies per cell. Standard protocols for the introduction of transforming DNA employ biolistic DNA delivery or polyethylene glycol treatment. Genetically stable, transgenic plants are obtained by modification of the plastid genome by homologous recombination, followed by selection for the transformed genome copy by the expression of marker genes that protect the cells from selective agents. Commonly used selective agents are antibiotics, including spectinomycin, streptomycin, kanamycin and chloramphenicol. Selection for resistance to amino acid analogues has also been successful. The types of plastid genome manipulations include gene deletion, gene insertion, and gene replacement, facilitated by specially designed transformation vectors. Methods are also available for post-transformation removal of marker genes. The model species for plastid genetic manipulation is Nicotiana tabacum, in which most protocols have been tested. Plastid transformation is also available in several solanaceous crops (tomato, potato, eggplant) and ornamental species (petunia, Nicotiana sylvestris). Significant progress has been made with Brasssicaceae including cabbage, oilseed rape and Arabidopsis. Recent additions to the crops in which plastid transformation is reproducibly obtained are lettuce, soybean and sugar beet. The monocots are a taxonomic group recalcitrant to plastid transformation; initial inroads have been made only in rice.
The genes encoding the 9 kDa and 4 kDa polypeptides of cytochrome b-559 have been located in pea chloroplast DNA by coupled transcription-translation of cloned restriction fragments of chloroplast DNA in a cell-free extract of Escherichia coli and by nucleotide sequence analysis. The genes (psbE and psbF) are located approximately 1.0 kbp downstream of the gene for cytochrome f and are transcribed in the opposite direction, similar to the arrangement in the chloroplast genomes of other higher plants. Nucleotide sequence analysis of this region revealed four open reading frames encoding hydrophobic proteins of 83 (psbE), 39 (psbF), 38 and 40 amino acid residues, which are co-transcribed as a single major RNA of 1.1 kb. The 5' and 3' ends of this RNA have been located by primer extension and S1 nuclease mapping. The 5' end of the RNA is located 140 bp upstream of the initiating ATG codon of psbE and is preceded by typical chloroplast promoter sequences. The 3' end of the RNA is located approximately 515 bp downstream of the TAA stop codon of psbF close to a stable stem-loop structure.
We have examined the function of inverted repeat sequences found at the 3' ends of plastid DNA transcription units in higher plants, using a homologous in vitro transcription extract. The inverted repeat sequences are ineffective as transcription terminators, but serve as efficient RNA processing elements. Synthetic RNAs are processed in a 3'-5' direction by a nuclease activity present in the transcription extract, generating nearly homogeneous 3' ends distal to the inverted repeat sequence. S1 nuclease protection experiments demonstrate that the 3' ends generated in vitro coincide with those found for plastid mRNAs in vivo. RNA molecules possessing inverted repeats near their 3' ends are substantially more stable than control RNAs in the chloroplast extract, and kinetic measurements indicate that each RNA has a unique decay rate. Coupled with previously published information suggesting that the differential accumulation of plastid RNAs during development is effectively controlled by post-transcriptional mechanisms, these results raise the possibility that RNA processing and stability, specifically involving 3' end inverted repeats, are important regulatory features of plastid gene expression.
The gene for the large subunit (LS) of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCase/Oase) from tobacco has been cloned in pBR322 and sequenced. The coding region contains 1431 bp (477 codons). The deduced amino acid sequence of tobacco LS protein shows 90% homology with those of maize and spinach LS. The positions in the gene corresponding to the 5' and the 3' ends of tobacco LS mRNA have been located on the DNA sequence by the S1 nuclease mapping procedure. The LS gene promoter sequence has homology with Escherichia coli promoter sequences; its terminator sequence is capable of forming a stem-and-loop structure. A sequence GGAGG, which is complementary to a sequence near the 3' end of tobacco chloroplast 16S rRNA and a putative ribosome binding site, occurs 6-10 bp upstream from the initiation codon.
We report here a 100-fold increased frequency of plastid transformation in tobacco by selection for a chimeric aadA gene encoding aminoglycoside 3"-adenylyltransferase, as compared with that obtained with mutant 16S rRNA genes. Expression of aadA confers resistance to spectinomycin and streptomycin. In transforming plasmid pZS197, a chimeric aadA is cloned between rbcL and open reading frame ORF512 plastid gene sequences. Selection was for spectinomycin resistance after biolistic delivery of pZS197 DNA into leaf cells. DNA gel-blot analysis confirmed incorporation of the chimeric aadA gene into the plastid genome by two homologous recombination events via the flanking plastid gene sequences. The chimeric gene became homoplasmic in the recipient cells and is uniformly transmitted to the maternal seed progeny. The ability to transform routinely plastids of land plants opens the way to manipulate the process of photosynthesis and to incorporate novel genes into the plastid genome of crops.
The chloroplast genome consists of homogeneous circular DNA molecules. To date, the entire nucleotide sequences (120-190 kbp) of chloroplast genomes have been determined from eight plant species. The chloroplast genomes of land plants and green algae contain about 110 different genes, which can be classified into two main groups: genes involved in gene expression and those related to photosynthesis. The red alga Porphyra chloroplast genome has 70 additional genes, one-third of which are related to biosynthesis of amino acids and other low molecular mass compounds. Chloroplast genes contain at least three structurally distinct promoters and transcribe two or more classes of RNA polymerase. Two chloroplast genes, rps12 of land plants and psaA of Chlamydomonas, are divided into two to three pieces and scattered over the genome. Each portion is transcribed separately, and two to three separate transcripts are joined together to yield a functional mRNA by trans-splicing. RNA editing (C to U base changes) occurs in some of the chloroplast transcripts. Most edited codons are functionally significant, creating start and stop codons and changing codons to retain conserved amino acids.