Phytosensors: harnessing plants to understand the world around us.

Although plants are sessile, their ubiquitous distribution, ability to harness energy from the sun, and ability to sense above and belowground signals make them ideal candidates for biosensor development. Synthetic biology has allowed scientists to reimagine biosensors as engineered devices that are focused on accomplishing novel tasks. As such, a new wave of plant-based sensors, phytosensors, are being engineered as multi-component sense-and-report devices that can alert human operators to a variety of hazards. While phytosensors are intrinsically tied to agriculture, a new generation of phytosensors has been envisioned to function in the built environment and even in austere environments, such as space. In this review, we will explore the current state of the art with regard to phytosensor engineering.

Structures and interactions forming stable shellac-casein nanocomplexes with a pH-cycle.

Casein forms diverse structures with functionalities tunable by complexation with surfactants, and shellac is an emerging surfactant. In the present work, molecular and mesoscopic structures of shellac and micellar casein and the underlying interactions after treatment with a pH-cycle were investigated. Dispersions with 0.5 % w/v shellac and various shellac:casein mass ratios were prepared at pH 12.0 to dissolve shellac and dissociate casein micelles, followed by neutralization to pH 7.0 to form complexes. Both covalent and non-covalent (hydrogen bonding, electrostatic, and hydrophobic) interactions contributed to the complex formation. The formed complexes had an average diameter of ~80 nm. The complexation of shellac and casein prevented the precipitation of protonated shellac during neutralization, and dispersions with casein:shellac mass ratios of 2:1 and above were absent of precipitates at pH 7.0. The formed nanocomplexes may have applications for preparing novel colloidal systems and loading lipophilic bioactive compounds.

Aqueous Ozone Exposure Inhibits Sporulation in the Cyclospora cayetanensis Surrogate Eimeria acervulina.

Ozone is a potent disinfecting agent used to treat potable water and wastewater, effectively clearing protozoa such as Giardia and Cryptosporidium spp. It is unclear whether ozone treatment of water or fresh produce can reduce the spread of the emerging parasite Cyclospora cayetanensis, which causes cyclosporiasis in humans. Obtaining viable C. cayetanensis oocysts to evaluate inactivation methods is challenging because we lack the means to propagate them in vitro, because of delays in case reporting, and because health departments typically add inactivating fixatives to clinical specimens. Research in various surrogate organisms has sought to bolster understanding of the biology of C. cayetanensis. Among these surrogates is the poultry parasite Eimeria acervulina, a closely related and easily cultured parasite of economic significance. We used this surrogate to evaluate the consequences of ozone treatment, using the sporulation state as an indicator of infectious potential. Treating with ozonated water acidified with citric acid reduced sporulation ability in a dose-dependent manner; treatment with up to 4.93 mg/L initial concentration of ozone resulted in a 93% inactivation of sporulation by 7 days posttreatment. This developmental arrest was accompanied by transcriptional changes in genes involved in regulating the response to reactive oxygen species (ROS) in a time course that is consistent with the production of oxygen free radicals. This study shows that ozone is highly effective in preventing sporulation of E. acervulina, a model coccidian used as a surrogate for Cyclospora. Furthermore, ozone exposure induced molecular responses to general oxidative stress, documented with several well-characterized antioxidant enzymes.

Synthetic microbe-to-plant communication channels.

Plants and microbes communicate to collaborate to stop pests, scavenge nutrients, and react to environmental change. Microbiota consisting of thousands of species interact with each other and plants using a large chemical language that is interpreted by complex regulatory networks. In this work, we develop modular interkingdom communication channels, enabling bacteria to convey environmental stimuli to plants. We introduce a "sender device" in Pseudomonas putida and Klebsiella pneumoniae, that produces the small molecule p-coumaroyl-homoserine lactone (pC-HSL) when the output of a sensor or circuit turns on. This molecule triggers a "receiver device" in the plant to activate gene expression. We validate this system in Arabidopsis thaliana and Solanum tuberosum (potato) grown hydroponically and in soil, demonstrating its modularity by swapping bacteria that process different stimuli, including IPTG, aTc and arsenic. Programmable communication channels between bacteria and plants will enable microbial sentinels to transmit information to crops and provide the building blocks for designing artificial consortia.

AI to enable plant cell metabolic engineering.

Plant metabolic engineering must take into consideration the heterogeneous cell types that play a role in metabolite production; cells do not participate equally. We posit that artificial intelligence (AI) developed for biomedical purposes can be applied to plant cell characterization to accelerate the development of metabolic engineering strategies in plants.

Development of new binary expression systems for plant synthetic biology.

KEY MESSAGE: A novel plant binary expression system was developed from the compactin biosynthetic pathway 27 of Penicillium citrinum ML-236B. The system achieved >fivefold activation of gene expression in 28 transgenic tobacco. A diverse and well-characterized genetic toolset is fundamental to achieve the overall goals of plant synthetic biology. To properly coordinate expression of a multigene pathway, this toolset should include binary systems that control gene expression at the level of transcription. In plants, few highly functional, orthogonal transcriptional regulators have been identified. Here, we describe the process of developing synthetic plant transcription factors using regulatory elements from the Penicillium citrinum ML-236B (compactin) pathway. This pathway contains several genes including mlcA and mlcC that are transcriptionally regulated in a dose-dependent manner by the activator mlcR. In Nicotiana benthamiana, we first expressed mlcR with several cognate synthetic promoters driving expression of GFP. Synthetic promoters contained operator sequences from the compactin gene cluster. Following identification of the most active synthetic promoter, the DNA-binding domain from mlcR was used to generate chimeric transcription factors containing variable activation domains, including QF from the Neurospora crassa Q-system. Activity was measured at both protein and RNA levels which correlated with an R2 value of 0.94. A synthetic transcription factor with a QF activation domain increased gene expression from its synthetic promoter up to sixfold in N. benthamiana. Two systems were characterized in transgenic tobacco plants. The QF-based plants maintained high expression in tobacco, increasing expression from the cognate synthetic promoter by fivefold. Transgenic plants and non-transgenic plants were morphologically indistinguishable. The framework of this study can easily be adopted for other putative transcription factors to continue improvement of the plant synthetic biology toolbox.

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.

Soil elemental changes during human decomposition.

Mammalian decomposition provides pulses of organic matter to the local ecosystem creating ephemeral hotspots of nutrient cycling. While changes to soil biogeochemistry in these hotspots have been described for C and N, patterns associated with deposition and cycling of other elements have not received the same attention. The goal of our study was to evaluate temporal changes to a broad suite of dissolved elements in soils impacted by human decomposition on the soil surface including: 1) abundant mineral elements in the human body (K, Na, S, P, Ca, and Mg), 2) trace elements in the human body (Fe, Mn, Se, Zn, Cu, Co, and B), and 3) Al which is transient in the human body but common in soils. We performed a four-month human decomposition trial at the University of Tennessee Anthropology Research Facility and quantified elemental concentrations dissolved in the soil solution, targeting the mobile and bioavailable fraction. We identified three groups of elements based on their temporal patterns. Group 1 elements appeared to be cadaver-derived (Na, K, P, S) and their persistence in soil varied based upon soluble organic forms (P), the dynamics of the soil exchange complex (Na, K), and gradual releases attributable to microbial degradation (S). Group 2 elements (Ca, Mg, Mn, Se, B) included three elements that have greater concentrations in soil than would be expected based on cadaver inputs alone, suggesting that these elements partially originate from the soil exchange (Ca, Mg), or are solubilized as a result of soil acidification (Mn). Group 3 elements (Fe, Cu, Zn, Co, Al) increased late in the decomposition process, suggesting a gradual solubilization from soil minerals under acidic pH conditions. This work presents a detailed longitudinal characterization of changes in dissolved soil elements during human decomposition furthering our understanding of elemental deposition and cycling in these environments.

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.

Shellac Micelles Loaded with Curcumin Using a pH Cycle to Improve Dispersibility, Bioaccessibility, and Potential for Colon Delivery.

Delivery systems smaller than 50 nm are advantageous for cancer prevention. In this study, curcumin was dissolved in shellac micelles following co-dissolving at pH 13.0 and neutralization using glucono-delta-lactone. With 5% w/v shellac and 0.5-5 mg/mL curcumin, the loading capacity and encapsulation efficiency were up to 8.0 and 92.6%, respectively, and the nanocapsules had an average diameter of 20 nm. Differential scanning calorimetry, FTIR spectroscopy, and fluorescence spectroscopy results confirmed the encapsulation of curcumin in an amorphous state in shellac micelles. The neutral nanocapsule dispersions maintained the particle dimension and had less than 10% curcumin degradation during 4 week storage at 4 °C. Nanoencapsulating curcumin enhanced in vitro bioavailability and antiproliferation activity against colon cancer cells. After simulated digestions, ∼60% of the nanoencapsulated curcumin was not available for intestinal absorption, nanocapsules retained their structure, and nanoencapsulated curcumin remained active against colon cancer cells, indicating the potential delivery for colorectal cancer prevention.

Duckweed: a potential phytosensor for heavy metals.

Globally, heavy metal (HM) contamination is one of the primary causes of environmental pollution leading to decreased quality of life for those affected. In particular, HM contamination in groundwater poses a serious risk to human health and the potential for destabilization of aquatic ecosystems. At present, strategies to remove HM contamination from wastewater are inefficient, costly, laborious, and often the removal poses as much risk to the environment as the initial contamination. Phytoremediation, plant-based removal of contaminants from soil or water, has long been viewed as an economical and sustainable solution to remove toxic metals from the environment. However, to date, phytoremediation has demonstrated limited successes despite a large volume of literature supporting its potential. A key aspect for achieving robust and meaningful phytoremediation is the selection of a plant species that is well suited to the task. For the removal of pollutants from wastewater, hydrophytes, like duckweed, exhibit significant potential due to their rapid growth on nutrient-rich water, ease of collection, and ability to survive in various ecosystems. As a model for ecotoxicity studies, duckweed is an ideal candidate, as it is easy to cultivate under controlled and even sterile conditions, and the rapid growth enables multi-generational studies. Similarly, recent advances in the genetic engineering and genome-editing of duckweed will enable the transition from fundamental ecotoxicity studies to engineered solutions for phytoremediation of HMs. This review will provide insight into the suitability of duckweeds for phytoremediation of HMs and strategies for engineering next-generation duckweed to provide real-world environmental solutions.

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.

Specific Bacterial Pathogen Phytosensing Is Enabled by a Synthetic Promoter-Transcription Factor System in Potato.

Phytosensors are genetically engineered plant-based sensors that feature synthetic promoters fused to reporter genes to sense and report the presence of specific biotic and abiotic stressors on plants. However, when induced reporter gene output is below detectable limits, owing to relatively weak promoters, the phytosensor may not function as intended. Here, we show modifications to the system to amplify reporter gene signal by using a synthetic transcription factor gene driven by a plant pathogen-inducible synthetic promoter. The output signal was unambiguous green fluorescence when plants were infected by pathogenic bacteria. We produced and characterized a phytosensor with improved sensing to specific bacterial pathogens with targeted detection using spectral wavelengths specific to a fluorescence reporter at 3 m standoff detection. Previous attempts to create phytosensors revealed limitations in using innate plant promoters with low-inducible activity since they are not sufficient to produce a strong detectable fluorescence signal for standoff detection. To address this, we designed a pathogen-specific phytosensor using a synthetic promoter-transcription factor system: the S-Box cis-regulatory element which has low-inducible activity as a synthetic 4xS-Box promoter, and the Q-system transcription factor as an amplifier of reporter gene expression. This promoter-transcription factor system resulted in 6-fold amplification of the fluorescence after infection with a potato pathogen, which was detectable as early as 24 h post-bacterial infection. This novel bacterial pathogen-specific phytosensor potato plant demonstrates that the Q-system may be leveraged as a powerful orthogonal tool to amplify a relatively weak synthetic inducible promoter, enabling standoff detection of a previously undetectable fluorescence signal. Pathogen-specific phytosensors would be an important asset for real-time early detection of plant pathogens prior to the display of disease symptoms on crop 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.

Rational design and testing of abiotic stress-inducible synthetic promoters from poplar cis-regulatory elements.

Abiotic stress resistance traits may be especially crucial for sustainable production of bioenergy tree crops. Here, we show the performance of a set of rationally designed osmotic-related and salt stress-inducible synthetic promoters for use in hybrid poplar. De novo motif-detecting algorithms yielded 30 water-deficit (SD) and 34 salt stress (SS) candidate DNA motifs from relevant poplar transcriptomes. We selected three conserved water-deficit stress motifs (SD18, SD13 and SD9) found in 16 co-expressed gene promoters, and we discovered a well-conserved motif for salt response (SS16). We characterized several native poplar stress-inducible promoters to enable comparisons with our synthetic promoters. Fifteen synthetic promoters were designed using various SD and SS subdomains, in which heptameric repeats of five-to-eight subdomain bases were fused to a common core promoter downstream, which, in turn, drove a green fluorescent protein (GFP) gene for reporter assays. These 15 synthetic promoters were screened by transient expression assays in poplar leaf mesophyll protoplasts and agroinfiltrated Nicotiana benthamiana leaves under osmotic stress conditions. Twelve synthetic promoters were induced in transient expression assays with a GFP readout. Of these, five promoters (SD18-1, SD9-2, SS16-1, SS16-2 and SS16-3) endowed higher inducibility under osmotic stress conditions than native promoters. These five synthetic promoters were stably transformed into Arabidopsis thaliana to study inducibility in whole plants. Herein, SD18-1 and SD9-2 were induced by water-deficit stress, whereas SS16-1, SS16-2 and SS16-3 were induced by salt stress. The synthetic biology design pipeline resulted in five synthetic promoters that outperformed endogenous promoters in transgenic 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.