Gene introduction approaches in chloroplast transformation and its applications

Background Chloroplast is a type of plastid that is believed to be originated from ancestral cyanobacteria. Chloroplast besides being a major component for photosynthesis, also takes part in another major plant metabolism, making it one of the major components of plants. Main body Chloroplast transformation is an alternative and better genetic engineering approach compared to the nuclear transformation that has been widely applied in plant genetic engineering. Chloroplast transformation has exhibited various positive effects as compared to nuclear transformation. This is a more preferred technique by researchers. To carry out chloroplast transformation, the vector design must be performed, and a selectable marker needs to be incorporated before the chloroplast could uptake the construct. The common way of introducing a gene into the host, which is the chloroplast, involves the biolistic, PEG-mediated, carbon nanotubes carriers, UV-laser microbeam, and Agrobacterium-mediated transformation approaches. Apart from discussing the processes involved in introducing the gene into the chloroplast, this review also focuses on the various applications brought about by chloroplast transformation, particularly in the field of agriculture and environmental science. Conclusion Chloroplast transformation has shown a lot of advantages and proven to be a better alternative compared to nuclear genome transformation. Further studies must be conducted to uncover new knowledge regarding chloroplast transformation as well as to discover its additional applications in the fields of biotechnology.

Polymorphism in the Chloroplast ATP Synthase Beta-Subunit Is Associated with a Maternally Inherited Enhanced Cold Recovery in Cucumber

Cucumber (Cucumis sativus L.) is a warm-season crop that is sensitive to chilling temperatures and a maternally inherited cold tolerance exists in the heirloom cultivar ‘Chipper’ (CH). Because the organelles of cucumber show differential transmission (maternal for chloroplast and paternal for mitochondrion), this cold tolerance is hypothesized to be chloroplast-associated. The goal of this research was to characterize the cold tolerant phenotype from CH and determine its genetic basis. Doubled haploid (DH) lines were produced from CH and cold susceptible cucumbers, reciprocal hybrids with identical nuclear genotypes were produced, and plants were subjected to cold treatments under lights at 4 °C for 5.5 h. Hybrid plants with CH as the maternal parent had significantly higher fresh and dry weights 14 days after cold treatment compared to the reciprocal hybrid, revealing an enhanced cold recovery phenotype maternally conferred by CH. Results from analyses of the nuclear transcriptome and reactive oxygen species (ROS) between reciprocal hybrids were consistent with the cold recovery phenotype. Sequencing of the chloroplast genome and transcriptome of the DH parents and reciprocal hybrids, respectively, revealed one maternally transmitted non-synonymous single nucleotide polymorphism (SNP) in the chloroplast F1FO-ATP synthase (CF1FO-ATPase) beta-subunit gene (atpB) of CH which confers an amino acid change from threonine to arginine. Protein modeling revealed that this change is located at the interface of the alpha- and beta-subunits in the CF1FO-ATPase complex. Polymorphisms in the CF1FO-ATPase complex have been associated with stress tolerances in other plants, and selection for or creation of polymorphic beta-subunit proteins by chloroplast transformation or gene editing could condition improved recovery from cold stress in plants.

Characterization of the Chloroplast Genome Facilitated the Transformation of Parachlorella kessleri-I, A Potential Marine Alga for Biofuel Production

Introduction: The microalga Parachlorella kessleri-I produces high biomass and lipid content that could be suitable for producing economically viable biofuel at a commercial scale. Sequencing the complete chloroplast genome is crucial for the construction of a species-specific chloroplast transformation vector. Methods: In this study, the complete chloroplast genome sequence (cpDNA) of P. kessleri-I was assembled; annotated and genetic transformation of the chloroplast was optimized. For the chloroplast transformation, we have tested two antibiotic resistance makers, aminoglycoside adenine transferase (aadA) gene and Sh-ble gene conferring resistance to spectinomycin and zeocin, respectively. Transgene integration and homoplasty determination were confirmed using PCR, Southern blot and Droplet Digital PCR. Results: The chloroplast genome (109,642 bp) exhibited a quadripartite structure with two reverse repeat regions (IRA and IRB), a long single copy (LSC), and a small single copy (SSC) region. The genome encodes 116 genes, with 80 protein-coding genes, 32 tRNAs and 4 rRNAs. The cpDNA provided essential information like codons, UTRs and flank sequences for homologous recombination to make a species-specific vector that facilitated the transformation of P. kessleri-I chloroplast. The transgenic algal colonies were retrieved on a TAP medium containing 400 mg. L-1 spectinomycin, but no transgenic was recovered on the zeocin-supplemented medium. PCR and Southern blot analysis ascertained the transgene integration into the chloroplast genome, via homologous recombination. The chloroplast genome copy number in wildtype and transgenic P. kessleri-I was determined using Droplet Digital PCR. Conclusion: The optimization of stable chloroplast transformation in marine alga P. kessleri-I should open a gateway for directly engineering the strain for carbon concentration mechanisms to fix more CO2, improving the photosynthetic efficiency and reducing the overall biofuels production cost.

Neither the availability of D2 nor CP43 limits the biogenesis of PSII in tobacco

The pathway of photosystem II assembly is well understood and multiple auxiliary proteins supporting it have been identified. By contrast, little is known about rate-limiting steps controlling PSII biogenesis. In the green alga Chlamydomonas reinhardtii, biosynthesis of the chloroplast-encoded D2 reaction center subunit (PsbD) limits PSII accumulation. To determine the importance of D2 synthesis for PSII accumulation in vascular plants and elucidate the contributions of transcriptional and translational regulation, the 5’-untranslated region of psbD was modified via chloroplast transformation in tobacco. A drastic reduction in psbD mRNA abundance resulted in a strong decrease of PSII content, impaired photosynthetic electron transport, and retarded growth under autotrophic conditions. Overexpression of the psbD mRNA also increased transcript abundance of psbC (the CP43 inner antenna protein), which is co-transcribed with psbD. Because translation efficiency remained unaltered, translation output of pbsD and psbC increased with mRNA abundance. However, this did not result in increased PSII accumulation. The introduction of point mutations into the Shine-Dalgarno-like sequence or start codon of psbD decreased translation efficiency without causing pronounced effects on PSII accumulation and function. These data show that neither transcription nor translation of psbD and psbC are rate-limiting for PSII biogenesis in vascular plants, and that PSII assembly and accumulation in tobacco are controlled by different mechanisms than in Chlamydomonas.One sentence summaryPSII biogenesis in tobacco is neither limited by transcript accumulation nor translation of psbD and psbC.

Improvement of Chloroplast Transformation Using CRISPR/Cas9

Chloroplasts are organelles that contain genetic materials (DNA) in higher plant cells. The special genetic characteristics of chloroplasts mean that plasmid transformation has important research value, so it has become an important research direction second to nuclear transformation. Although the techniques of chloroplast genome modification have been successfully applied in tobacco and extended to other high plants, there are still many limitations. Exogenous genes are integrated into the chloroplast genome through homologous recombination. Therefore, the low efficiency of homologous recombination directly limits transformation efficiency. Gene editing with fixed-point cutting function and DNA damage repair mechanism may effectively improve the efficiency. In the present study, we aimed to use CRISPR/Cas9 to cut the site between two homologous recombinant fragments in chloroplast transformation to improve the efficiency by activating the DNA damage repair mechanism. The Cas9 gene and gRNA were added to the chloroplast transformation system of tobacco by co-transformation or integration into a transformation vector. The acquired resistant plants were screened by multiple selection of spectinomycin and chloroplast DNA was isolated for molecular detection by PCR. The results showed that the efficiency of chloroplast transformation increased by 6–10 times with the addition of gene editing technology. Although the transformation efficiency was still far below the level of nuclear transformation, this study may help to increase the efficiency of the plant chloroplast transformation system, and expand the types of plant receptors.

A Phosphite Dehydrogenase Variant with Promiscuous Access to Nicotinamide Cofactor Pools Sustains Fast Phosphite-Dependent Growth of Transplastomic Chlamydomonas reinhardtii

Heterologous expression of the NAD+-dependent phosphite dehydrogenase (PTXD) bacterial enzyme from Pseudomonas stutzerii enables selective growth of transgenic organisms by using phosphite as sole phosphorous source. Combining phosphite fertilization with nuclear expression of the ptxD transgene was shown to be an alternative to herbicides in controlling weeds and contamination of algal cultures. Chloroplast expression of ptxD in Chlamydomonas reinhardtii was proposed as an environmentally friendly alternative to antibiotic resistance genes for plastid transformation. However, PTXD activity in the chloroplast is low, possibly due to the low NAD+/NADP+ ratio, limiting the efficiency of phosphite assimilation. We addressed the intrinsic constraints of the PTXD activity in the chloroplast and improved its catalytic efficiency in vivo via rational mutagenesis of key residues involved in cofactor binding. Transplastomic lines carrying a mutagenized PTXD version promiscuously used NADP+ and NAD+ for converting phosphite into phosphate and grew faster compared to those expressing the wild type protein. The modified PTXD enzyme also enabled faster and reproducible selection of transplastomic colonies by directly plating on phosphite-containing medium. These results allow using phosphite as selective agent for chloroplast transformation and for controlling biological contaminants when expressing heterologous proteins in algal chloroplasts, without compromising on culture performance.