Generation, analysis, and transformation of macro-chloroplast Potato (Solanum tuberosum) lines for chloroplast biotechnology.

Chloroplast biotechnology is a route for novel crop metabolic engineering. The potential bio-confinement of transgenes, the high protein expression and the possibility to organize genes into operons represent considerable advantages that make chloroplasts valuable targets in agricultural biotechnology. In the last 3 decades, chloroplast genomes from a few economically important crops have been successfully transformed. The main bottlenecks that prevent efficient transformation in a greater number of crops include the dearth of proven selectable marker gene-selection combinations and tissue culture methods for efficient regeneration of transplastomic plants. The prospects of increasing organelle size are attractive from several perspectives, including an increase in the surface area of potential targets. As a proof-of-concept, we generated Solanum tuberosum (potato) macro-chloroplast lines overexpressing the tubulin-like GTPase protein gene FtsZ1 from Arabidopsis thaliana. Macro-chloroplast lines exhibited delayed growth at anthesis; however, at the time of harvest there was no significant difference in height between macro-chloroplast and wild-type lines. Macro-chloroplasts were successfully transformed by biolistic DNA-delivery and efficiently regenerated into homoplasmic transplastomic lines. We also demonstrated that macro-chloroplasts accumulate the same amount of heterologous protein than wild-type organelles, confirming efficient usage in plastid engineering. Advantages and limitations of using enlarge compartments in chloroplast biotechnology are discussed.

MoChlo: A Versatile, Modular Cloning Toolbox for Chloroplast Biotechnology.

Plant synthetic biology is a rapidly evolving field with new tools constantly emerging to drive innovation. Of particular interest is the application of synthetic biology to chloroplast biotechnology to generate plants capable of producing new metabolites, vaccines, biofuels, and high-value chemicals. Progress made in the assembly of large DNA molecules, composing multiple transcriptional units, has significantly aided in the ability to rapidly construct novel vectors for genetic engineering. In particular, Golden Gate assembly has provided a facile molecular tool for standardized assembly of synthetic genetic elements into larger DNA constructs. In this work, a complete modular chloroplast cloning system, MoChlo, was developed and validated for fast and flexible chloroplast engineering in plants. A library of 128 standardized chloroplast-specific parts (47 promoters, 38 5' untranslated regions [5'UTRs], nine promoter:5'UTR fusions, 10 3'UTRs, 14 genes of interest, and 10 chloroplast-specific destination vectors) were mined from the literature and modified for use in MoChlo assembly, along with chloroplast-specific destination vectors. The strategy was validated by assembling synthetic operons of various sizes and determining the efficiency of assembly. This method was successfully used to generate chloroplast transformation vectors containing up to seven transcriptional units in a single vector (∼10.6-kb synthetic operon). To enable researchers with limited resources to engage in chloroplast biotechnology, and to accelerate progress in the field, the entire kit, as described, is available through Addgene at minimal cost. Thus, the MoChlo kit represents a valuable tool for fast and flexible design of heterologous metabolic pathways for plastid metabolic engineering.

Identification, characterization and expression analysis of MicroRNAs and their targets in the potato (Solanum tuberosum).

MicroRNAs (miRNAs) are recognized as a class of important post-transcriptional expression regulators that act on their target genes by degradation of target mRNAs or by inhibition of target protein translation. Compared with the current numbers of identified miRNAs for other species in the plant kingdom, a large number of potential miRNAs remains to be identified in potato. In this study, using a newly modified comparative genome strategy, a total of 202 potential potato miRNAs were identified, which belong to 78 families. miR162, miR167, and miR396 are highly expressed in all tested organs. miR372 is highly expressed in flowers. A total of 1094 miRNA targets were predicted and some of them encode transcription factors as well as genes that function in stress response, signal transduction, and a variety of other metabolic processes. Gene ontology (GO) analysis implicates that these targets are involved in 545 biological processes. Of those processes, 28 are related to potato defense mechanisms against bacteria, viruses, and fungi, the metabolism of molecules such as carbon, sucrose, starch, and lipid, and the development of primary and lateral roots. Pathway enrichment analysis, based on the Kyoto Encyclopedia of Genes and Genomes (KEGG), demonstrates that the identified miRNAs participated in 98 metabolism networks, some of which include sucrose metabolism, fatty acid metabolism, amino acid metabolism, carbon fixation, and the biosynthesis of plant hormones.