Heterologous Expression of OtsB Increases Tuber Yield and Phenotypic Stability in Potato under Both Abiotic and Biotic Stresses.

Climate-smart and sustainable crops are needed for the future. Engineering crops for tolerance of both abiotic and biotic stress is one approach. The accumulation of trehalose, controlled through trehalose-6-phosphate synthase (TPS) or OtsA and trehalose-6-phosphate phosphatase (TPP) or OtsB genes in microbes, is known to provide protection for many microbial and fungal species against abiotic stress. The effect of trehalose accumulation in plant species is less understood. Here, we studied the heterologous expression of Escherichia coli OtsB in potato (Solanum tuberosum var. 'Desiree') with regards to stress tolerance. The performance of transgenic lines was assessed in both growth chambers and greenhouse mesocosms. Overexpressing potato OtsB lines significantly increased resilience to heat, photoperiod, herbivory, and competition when compared with wildtype plants. Most strikingly, when subjected to high temperatures, transgenic lines exhibited a significantly lower reduction in tuber yield ranging from 40% to 77%, while wildtype plants experienced a 95% decrease in tuber yield. When exposed to competitors in a selected StSP3D::OtsB line, tuber yield was 1.6 times higher than wildtype. Furthermore, transgenic lines performed significantly better under low-nutrient regimes: under competition, yield increased by 1.5-fold. Together, these results demonstrate that increased trehalose has the potential to create more resistant and stable crop plants.

Plastid engineering using episomal DNA.

KEY MESSAGE: Novel episomal systems have the potential to accelerate plastid genetic engineering for application in plant synthetic biology. Plastids represent valuable subcellular compartments for genetic engineering of plants with intrinsic advantages to engineering the nucleus. The ability to perform site-specific transgene integration by homologous recombination (HR), coordination of transgene expression in operons, and high production of heterologous proteins, all make plastids an attractive target for synthetic biology. Typically, plastid engineering is performed by homologous recombination; however, episomal-replicating vectors have the potential to accelerate the design/build/test cycles for plastid engineering. By accelerating the timeline from design to validation, it will be possible to generate translational breakthroughs in fields ranging from agriculture to biopharmaceuticals. Episomal-based plastid engineering will allow precise single step metabolic engineering in plants enabling the installation of complex synthetic circuits with the ambitious goal of reaching similar efficiency and flexibility of to the state-of-the-art genetic engineering of prokaryotic systems. The prospect to design novel episomal systems for production of transplastomic marker-free plants will also improve biosafety for eventual release in agriculture.

Engineered gamma radiation phytosensors for environmental monitoring.

Nuclear energy, already a practical solution for supplying energy on a scale similar to fossil fuels, will likely increase its footprint over the next several decades to meet current climate goals. Gamma radiation is produced during fission in existing nuclear reactors and thus the need to detect leakage from nuclear plants, and effects of such leakage on ecosystems will likely also increase. At present, gamma radiation is detected using mechanical sensors that have several drawbacks, including: (i) limited availability; (ii) reliance on power supply; and (iii) requirement of human presence in dangerous areas. To overcome these limitations, we have developed a plant biosensor (phytosensor) to detect low-dose ionizing radiation. The system utilizes synthetic biology to engineer a dosimetric switch into potato utilizing the plant's native DNA damage response (DDR) machinery to produce a fluorescent output. In this work, the radiation phytosensor was shown to respond to a wide range of gamma radiation exposure (10-80 Grey) producing a reporter signal that was detectable at >3 m. Further, a pressure test of the top radiation phytosensor in a complex mesocosm demonstrated full function of the system in a 'real world' scenario.

Genome-Editing of FtsZ1 for Alteration of Starch Granule Size in Potato Tubers.

Genome-editing has enabled rapid improvement for staple food crops, such as potato, a key beneficiary of the technology. In potato, starch contained within tubers represents the primary product for use in food and non-food industries. Starch granules are produced in the plastids of tubers with plastid size correlated with the size of starch grana. The division of plastids is controlled by proteins, including the tubulin-like GTPase FtsZ1. The altered expression of FtsZ1 has been shown to disrupt plastid division, leading to the production of "macro-plastid"-containing plants. These macro-chloroplast plants are characterized by cells containing fewer and enlarged plastids. In this work, we utilize CRISPR/Cas9 to generate FtsZ1 edited potato lines to demonstrate that genome-editing can be used to increase the size of starch granules in tubers. Altered plastid morphology was comparable to the overexpression of FtsZ1 in previous work in potato and other crops. Several lines were generated with up to a 1.98-fold increase in starch granule size that was otherwise phenotypically indistinguishable from wild-type plants. Further, starch paste from one of the most promising lines showed a 2.07-fold increase in final viscosity. The advantages of enlarged starch granules and the potential of CRISPR/Cas9-based technologies for food crop improvement are further discussed.

Genetic Engineering of Potato (Solanum tuberosum) Chloroplasts Using the Small Synthetic Plastome "Mini-Synplastome".

In the rapidly expanding field of synthetic biology, chloroplasts represent attractive targets for installation of valuable genetic circuits in plant cells. Conventional methods for engineering the chloroplast genome (plastome) have relied on homologous recombination (HR) vectors for site-specific transgene integration for over 30 years. Recently, episomal-replicating vectors have emerged as valuable alternative tools for genetic engineering of chloroplasts. With regard to this technology, in this chapter we describe a method for engineering potato (Solanum tuberosum) chloroplasts to generate transgenic plants using the small synthetic plastome (mini-synplastome). In this method, the mini-synplastome is designed for Golden Gate cloning for easy assembly of chloroplast transgene operons. Mini-synplastomes have the potential to accelerate plant synthetic biology by enabling complex metabolic engineering in plants with similar flexibility of engineered microorganisms.

Building the Plant SynBio Toolbox through Combinatorial Analysis of DNA Regulatory Elements.

While the installation of complex genetic circuits in microorganisms is relatively routine, the synthetic biology toolbox is severely limited in plants. Of particular concern is the absence of combinatorial analysis of regulatory elements, the long design-build-test cycles associated with transgenic plant analysis, and a lack of naming standardization for cloning parts. Here, we use previously described plant regulatory elements to design, build, and test 91 transgene cassettes for relative expression strength. Constructs were transiently transfected into Nicotiana benthamiana leaves and expression of a fluorescent reporter was measured from plant canopies, leaves, and protoplasts isolated from transfected plants. As anticipated, a dynamic level of expression was achieved from the library, ranging from near undetectable for the weakest cassette to a ∼200-fold increase for the strongest. Analysis of expression levels in plant canopies, individual leaves, and protoplasts were correlated, indicating that any of the methods could be used to evaluate regulatory elements in plants. Through this effort, a well-curated 37-member part library of plant regulatory elements was characterized, providing the necessary data to standardize construct design for precision metabolic engineering in plants.

High-Throughput Transfection and Analysis of Soybean (Glycine max ) Protoplasts.

With the advent of plant synthetic biology, there is an urgent need to develop plant-based systems that are able to effectively enhance the speed of design-build-test cycles to screen large numbers of synthetic constructs. Thus far, protoplasts have served to fill this need, with cell suspension cultures serving as the primary source tissue to enable high-throughput protoplast experimentation. The possibility to use low-cost food-grade enzymes for cell wall digestion along with polyethylene glycol (PEG)-mediated transfection makes protoplasts particularly suited to automation and high-throughput screening. In other systems for which synthetic biology is well established (model bacteria and yeast), libraries of components, i.e., promoters, 5' untranslated regions, 3' untranslated regions, terminators, and transcription factors, serve as the basis for the design of complex genetic circuits. In order for synthetic biology to make similar strides in plant biology, well-characterized libraries of functional genetic parts for plants are required, necessitating the need for high-throughput protoplast assays.In this chapter, we describe an optimized method for the preparation of soybean (Glycine max ) dark-grown cell suspension cultures, followed by protoplast isolation, automated transfection , and subsequent screening.

Mini-synplastomes for plastid genetic engineering.

In the age of synthetic biology, plastid engineering requires a nimble platform to introduce novel synthetic circuits in plants. While effective for integrating relatively small constructs into the plastome, plastid engineering via homologous recombination of transgenes is over 30 years old. Here we show the design-build-test of a novel synthetic genome structure that does not disturb the native plastome: the 'mini-synplastome'. The mini-synplastome was inspired by dinoflagellate plastome organization, which is comprised of numerous minicircles residing in the plastid instead of a single organellar genome molecule. The first mini-synplastome in plants was developed in vitro to meet the following criteria: (i) episomal replication in plastids; (ii) facile cloning; (iii) predictable transgene expression in plastids; (iv) non-integration of vector sequences into the endogenous plastome; and (v) autonomous persistence in the plant over generations in the absence of exogenous selection pressure. Mini-synplastomes are anticipated to revolutionize chloroplast biotechnology, enable facile marker-free plastid engineering, and provide an unparalleled platform for one-step metabolic engineering in plants.

Imaging of multiple fluorescent proteins in canopies enables synthetic biology in plants.

Reverse genetics approaches have revolutionized plant biology and agriculture. Phenomics has the prospect of bridging plant phenotypes with genes, including transgenes, to transform agricultural fields. Genetically encoded fluorescent proteins (FPs) have revolutionized plant biology paradigms in gene expression, protein trafficking and plant physiology. While the first instance of plant canopy imaging of green fluorescent protein (GFP) was performed over 25 years ago, modern phenomics has largely ignored fluorescence as a transgene expression device despite the burgeoning FP colour palette available to plant biologists. Here, we show a new platform for stand-off imaging of plant canopies expressing a wide variety of FP genes. The platform-the fluorescence-inducing laser projector (FILP)-uses an ultra-low-noise camera to image a scene illuminated by compact diode lasers of various colours, coupled with emission filters to resolve individual FPs, to phenotype transgenic plants expressing FP genes. Each of the 20 FPs screened in plants were imaged at >3 m using FILP in a laboratory-based laser range. We also show that pairs of co-expressed fluorescence proteins can be imaged in canopies. The FILP system enabled a rapid synthetic promoter screen: starting from 2000 synthetic promoters transfected into protoplasts to FILP-imaged agroinfiltrated Nicotiana benthamiana plants in a matter of weeks, which was useful to characterize a water stress-inducible synthetic promoter. FILP canopy imaging was also accomplished for stably transformed GFP potato and in a split-GFP assay, which illustrates the flexibility of the instrument for analysing fluorescence signals in plant canopies.

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.

The Q-System as a Synthetic Transcriptional Regulator in Plants.

A primary focus of the rapidly growing field of plant synthetic biology is to develop technologies to precisely regulate gene expression and engineer complex genetic circuits into plant chassis. At present, there are few orthogonal tools available for effectively controlling gene expression in plants, with most researchers instead using a limited set of viral elements or truncated native promoters. A powerful repressible-and engineerable-binary system that has been repurposed in a variety of eukaryotic systems is the Q-system from Neurospora crassa. Here, we demonstrate the functionality of the Q-system in plants through transient expression in soybean (Glycine max) protoplasts and agroinfiltration in Nicotiana benthamiana leaves. Further, using functional variants of the QF transcriptional activator, it was possible to modulate the expression of reporter genes and to fully suppress the system through expression of the QS repressor. As a potential application for plant-based biosensors (phytosensors), we demonstrated the ability of the Q-system to amplify the signal from a weak promoter, enabling remote detection of a fluorescent reporter that was previously undetectable. In addition, we demonstrated that it was possible to coordinate the expression of multiple genes through the expression of a single QF activator. Based on the results from this study, the Q-system represents a powerful orthogonal tool for precise control of gene expression in plants, with envisioned applications in metabolic engineering, phytosensors, and biotic and abiotic stress tolerance.

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.

Visualization of RMRs (Receptor Membrane RING-H2) Dimerization in Nicotiana benthamiana Leaves Using a Bimolecular Fluorescence Complementation (BiFC) Assay.

The bimolecular fluorescent complementation (BiFC) is a fluorescent complementation method largely used to investigate protein-protein interaction in living cells. This method is based on the ability of two nonfluorescent fragments to assemble forming a native fluorescent reporter with the same spectral properties of the native reporter. Such fragments are fused to putative protein partners that in case of interaction will bring the two halves in close proximity, allowing for the reconstitution of an active fluorescent reporter. The BiFC has been used to investigate protein-protein interaction in a number of different organisms, including plants. In plant cells, many essential pathways of protein trafficking and subcellular localization necessitate the intervention of several protein units organized in multisubunit complexes. It is well known that vacuolar sorting of many secretory soluble proteins require the intervention of specific transmembrane cargo receptors able to interact forming dimers. In this chapter we describe a BiFC method for the efficient visualization of RMR (Receptor Membrane RING-H2) type 2 dimerization in agro-infiltrated Nicotiana benthamiana leaves. Furthermore, this relatively simple method represents an optimal strategy to test protein-protein interaction using any other putative protein partners of interest in plant cells.

RMR (Receptor Membrane RING-H2) type 1 and 2 show different promoter activities and subcellular localizations in Arabidopsis thaliana.

Soluble vacuolar proteins reach their compartments of final accumulation through the binding with specific transmembrane cargo receptors. In Arabidopsis thaliana two different families of receptors have been characterized. The AtVSRs (Vacuolar Sorting Receptor), which are known to be involved in the protein sorting to lytic vacuoles (LV), and the AtRMRs (Receptor Membrane RING-H2), for which there is less evidence for a role in the traffic to the protein storage vacuole (PSV). In this study we investigated the localization and tissue expression of two RMRs (AtRMR1 and 2) in their species of origin, A. thaliana. Our experiments using leaf protoplasts and transgenic plants supported previous results of subcellular localization in Nicotiana benthamiana that visualized AtRMR1 and 2 in the cisternae of endoplasmic reticulum (ER) and in the trans-Golgi network (TGN), respectively. The promoter activities of AtRMR1 and AtRMR2 detected in transgenic A. thaliana lines suggest that the expression of these two receptors only partially overlap in some organs and tissues. These results suggest that AtRMR1 and 2 are not functionally redundant, but could also interact and participate in the same cellular process in tissues with an overlapping expression.

Dimerization of the Vacuolar Receptors AtRMR1 and -2 from Arabidopsis thaliana Contributes to Their Localization in the trans-Golgi Network.

In Arabidopsis thaliana, different types of vacuolar receptors were discovered. The AtVSR (Vacuolar Sorting Receptor) receptors are well known to be involved in the traffic to lytic vacuole (LV), while few evidences demonstrate the involvement of the receptors from AtRMR family (Receptor Membrane RING-H2) in the traffic to the protein storage vacuole (PSV). In this study we focused on the localization of two members of AtRMR family, AtRMR1 and -2, and on the possible interaction between these two receptors in the plant secretory pathway. Our experiments with agroinfiltrated Nicotiana benthamiana leaves demonstrated that AtRMR1 was localized in the endoplasmic reticulum (ER), while AtRMR2 was targeted to the trans-Golgi network (TGN) due to the presence of a cytosolic 23-amino acid sequence linker. The fusion of this linker to an equivalent position in AtRMR1 targeted this receptor to the TGN, instead of the ER. By using a Bimolecular Fluorescent Complementation (BiFC) technique and experiments of co-localization, we demonstrated that AtRMR2 can make homodimers, and can also interact with AtRMR1 forming heterodimers that locate to the TGN. Such interaction studies strongly suggest that the transmembrane domain and the few amino acids surrounding it, including the sequence linker, are essential for dimerization. These results suggest a new model of AtRMR trafficking and dimerization in the plant secretory pathway.

Biochemical characterization of predicted Precambrian RuBisCO.

The antiquity and global abundance of the enzyme, RuBisCO, attests to the crucial and longstanding role it has played in the biogeochemical cycles of Earth over billions of years. The counterproductive oxygenase activity of RuBisCO has persisted over billions of years of evolution, despite its competition with the carboxylase activity necessary for carbon fixation, yet hypotheses regarding the selective pressures governing RuBisCO evolution have been limited to speculation. Here we report the resurrection and biochemical characterization of ancestral RuBisCOs, dating back to over one billion years ago (Gyr ago). Our findings provide an ancient point of reference revealing divergent evolutionary paths taken by eukaryotic homologues towards improved specificity for CO2, versus the evolutionary emphasis on increased rates of carboxylation observed in bacterial homologues. Consistent with these distinctions, in vivo analysis reveals the propensity of ancestral RuBisCO to be encapsulated into modern-day carboxysomes, bacterial organelles central to the cyanobacterial CO2 concentrating mechanism.

Transgenic tobacco plants with improved cyanobacterial Rubisco expression but no extra assembly factors grow at near wild-type rates if provided with elevated CO2.

Introducing a carbon-concentrating mechanism and a faster Rubisco enzyme from cyanobacteria into higher plant chloroplasts may improve photosynthetic performance by increasing the rate of CO2 fixation while decreasing losses caused by photorespiration. We previously demonstrated that tobacco plants grow photoautotrophically using Rubisco from Synechococcus elongatus, although the plants exhibited considerably slower growth than wild-type and required supplementary CO2 . Because of concerns that vascular plant assembly factors may not be adequate for assembly of a cyanobacterial Rubisco, prior transgenic plants included the cyanobacterial chaperone RbcX or the carboxysomal protein CcmM35. Here we show that neither RbcX nor CcmM35 is needed for assembly of active cyanobacterial Rubisco. Furthermore, by altering the gene regulatory sequences on the Rubisco transgenes, cyanobacterial Rubisco expression was enhanced and the transgenic plants grew at near wild-type growth rates, although still requiring elevated CO2 . We performed detailed kinetic characterization of the enzymes produced with and without the RbcX and CcmM35 cyanobacterial proteins. These transgenic plants exhibit photosynthetic characteristics that confirm the predicted benefits of introduction of non-native forms of Rubisco with higher carboxylation rate constants in vascular plants and the potential nitrogen-use efficiency that may be achieved provided that adequate CO2 is available near the enzyme.

A faster Rubisco with potential to increase photosynthesis in crops.

In photosynthetic organisms, D-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the major enzyme assimilating atmospheric CO2 into the biosphere. Owing to the wasteful oxygenase activity and slow turnover of Rubisco, the enzyme is among the most important targets for improving the photosynthetic efficiency of vascular plants. It has been anticipated that introducing the CO2-concentrating mechanism (CCM) from cyanobacteria into plants could enhance crop yield. However, the complex nature of Rubisco's assembly has made manipulation of the enzyme extremely challenging, and attempts to replace it in plants with the enzymes from cyanobacteria and red algae have not been successful. Here we report two transplastomic tobacco lines with functional Rubisco from the cyanobacterium Synechococcus elongatus PCC7942 (Se7942). We knocked out the native tobacco gene encoding the large subunit of Rubisco by inserting the large and small subunit genes of the Se7942 enzyme, in combination with either the corresponding Se7942 assembly chaperone, RbcX, or an internal carboxysomal protein, CcmM35, which incorporates three small subunit-like domains. Se7942 Rubisco and CcmM35 formed macromolecular complexes within the chloroplast stroma, mirroring an early step in the biogenesis of cyanobacterial ß-carboxysomes. Both transformed lines were photosynthetically competent, supporting autotrophic growth, and their respective forms of Rubisco had higher rates of CO2 fixation per unit of enzyme than the tobacco control. These transplastomic tobacco lines represent an important step towards improved photosynthesis in plants and will be valuable hosts for future addition of the remaining components of the cyanobacterial CCM, such as inorganic carbon transporters and the ß-carboxysome shell proteins.

ß-Carboxysomal proteins assemble into highly organized structures in Nicotiana chloroplasts.

The photosynthetic efficiency of C3 plants suffers from the reaction of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) with O2 instead of CO2 , leading to the costly process of photorespiration. Increasing the concentration of CO2 around Rubisco is a strategy used by photosynthetic prokaryotes such as cyanobacteria for more efficient incorporation of inorganic carbon. Engineering the cyanobacterial CO2 -concentrating mechanism, the carboxysome, into chloroplasts is an approach to enhance photosynthesis or to compartmentalize other biochemical reactions to confer new capabilities on transgenic plants. We have chosen to explore the possibility of producing ß-carboxysomes from Synechococcus elongatus PCC7942, a model freshwater cyanobacterium. Using the agroinfiltration technique, we have transiently expressed multiple ß-carboxysomal proteins (CcmK2, CcmM, CcmL, CcmO and CcmN) in Nicotiana benthamiana with fusions that target these proteins into chloroplasts, and that provide fluorescent labels for visualizing the resultant structures. By confocal and electron microscopic analysis, we have observed that the shell proteins of the ß-carboxysome are able to assemble in plant chloroplasts into highly organized assemblies resembling empty microcompartments. We demonstrate that a foreign protein can be targeted with a 17-amino-acid CcmN peptide to the shell proteins inside chloroplasts. Our experiments establish the feasibility of introducing carboxysomes into chloroplasts for the potential compartmentalization of Rubisco or other proteins.