Downloaded from http://cshperspectives.cshlp.org/ on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press Synthetic Botany Christian R. Boehm,1,4 Bernardo Pollak,1,4 Nuri Purswani,2 Nicola Patron,3 and Jim Haseloff1 1Department of Plant Sciences, University of Cambridge, Cambridge CB2 3EA, United Kingdom 2The IBM Place I, Singapore, 486072, Singapore 3Earlham Institute, Norwich NR4 7UG, United Kingdom Correspondence: [email protected] Plants are attractive platforms for synthetic biology and metabolic engineering. Plants’ modular and plastic body plans, capacity for photosynthesis, extensive secondary metabo- lism, and agronomic systems for large-scale production make them ideal targets for genetic reprogramming. However, efforts in this area have been constrained by slow growth, long life cycles, the requirement for specialized facilities, a paucity of efficient tools for genetic ma- nipulation, and the complexity of multicellularity. There is a need for better experimental and theoretical frameworks to understand the way genetic networks, cellular populations, and tissue-wide physical processes interact at different scales. We highlight new approaches to the DNA-based manipulation of plants and the use of advanced quantitative imaging techniques in simple plant models such as Marchantia polymorpha. These offer the prospects of improved understanding of plant dynamics and new approaches to rational engineering of plant traits. he development of new technologies for the crease genetic diversity in breeding populations Tproduction of larger and improved quanti- of food crops, which show lower allelic variation ties of goods from less feedstock has defined compared with wild populations. Selective human innovation for thousands of years, espe- breeding still plays a major role in the produc- cially in food production. To increase agricul- tion of new varieties but the emergence of mod- tural productivity, plant breeders have been se- ern plant biotechnology has led to a more tar- lecting for advantageous traits in crops since geted approach to increasing crop yields. Traits 9000–10,000 BC (Zohary et al. 2012). One no- of agricultural importance successfully intro- table success is the selective breeding of modern duced to plants using recombinant DNA tech- maize from teosinte (Beadle 1939; Doebley nology include herbicide resistance (Comai 2004), in which a handful of genetic differences et al. 1985), drought resistance (Kumar et al. caused substantial changes in ear morphology, 2014), pest resistance (Bates et al. 2005), path- kernel number, and crop yield. ogen resistance (Brunner et al. 2011; Horvath Technologies such as mutagenesis and in- et al. 2012; Jones et al. 2014), abiotic stress re- trogressive hybridization, developed in the early sistance (Jaglo-Ottosen et al. 1998), enhanced to mid-20th century, are commonly used to in- photosynthetic capacity (Ku et al. 2001), im- 4These authors contributed equally to this work. Editors: Daniel G. Gibson, Clyde A. Hutchison III, Hamilton O. Smith, and J. Craig Venter Additional Perspectives on Synthetic Biology available at www.cshperspectives.org Copyright # 2017 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a023887 Cite this article as Cold Spring Harb Perspect Biol 2017;9:a023887 1 Downloaded from http://cshperspectives.cshlp.org/ on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press C.R. Boehm et al. proved nitrogen use efficiency (Yanagisawa et al. the endogenous enhancer (Johnson et al. 2005; 2004), and added nutritional value (Ye et al. Laplaze et al. 2005; Gardner et al. 2009). 2000). However, although plants hold a unique In addition to stable nuclear transformation promise for bioproduction at the gigatonne mediated by Agrobacterium, transient methods scale, efforts in genetic engineering of plants have been developed for quantification of gene are lagging compared with microbial systems expression (Kapila et al. 1997) and bioproduc- (Antunes et al. 2009, 2011; Liu et al. 2011, tion. For instance, Nicotiana benthamiana leaves 2013; Koschmann et al. 2012; Wend et al. can be infiltrated with Agrobacterium at multiple 2013; Zurcher et al. 2013; Fethe et al. 2014; Mu¨l- regions of a single leaf and screened for gene ler et al. 2014). expression a few days after infection. This allows To exploit the unused potential of plants in relatively fast characterization of libraries of ge- expressing complex traits, we require (1) effi- netic components in a common genetic back- cient tools and methods for genetic engineer- ground (Sparkes et al. 2006; Engler et al. 2014; ing, (2) simpler multicellular plant chassis that Bru¨ckner et al. 2015). Transient leaf agroin- are amenable to rapid, high-throughput analy- filtration in Nicotiana has been adopted as sis, and (3) control over the biosynthesis, trans- an efficient method for the optimization of met- port, and storage of metabolites in specialized abolic pathways, such as artemisinin (van cells within complex plant tissues. In the follow- Herpen et al. 2010) and triterpene biosynthesis ing, we highlight progress made in these three pathways (reviewed in Thimmappa et al. 2014), key areas of concern for the future development and the bioproduction of vaccines (D’Aoust of plant synthetic biology. et al. 2008, Mardanova et al. 2015). Large invest- ments have been made in both the private and the public sectors to scale up production for NUCLEAR TRANSFORMATION IN PLANTS vaccines against viruses like Ebola (ZMapp, Implementation of synthetic biology in plants Mapp Biopharmaceuticals) and influenza calls for efficient methods for genetic manipu- (Medicago). Protoplasts, single cells derived lation. The most widely used technique for from tissues by digestion of cell walls, can also transformation of most plant species relies on be transformed transiently. Protoplast transfor- the native capacity of virulent strains of Agro- mation has been performed by electroporation bacterium tumefaciens to infect plant tissue and (Fromm et al. 1985; Ou-Lee et al. 1986; Haupt- to transfer a segment of its DNA (T-DNA) to mann et al. 1987; Negrutiu et al. 1987; Nishigu- the host cell (reviewed in Gelvin 2003). Because chi et al. 1987; Jones et al. 1989) or incubation in this method was adapted to allow delivery of a PEG solution (Krens et al. 1982; Potrykus et al. transgenes, it has been used extensively: thou- 1985), and is established as one of the preferred sands of transformants can be obtained in a methods for studying signaling pathways single experiment (Meyerowitz 1989; Ishizaki (reviewed in Sheen 2001). Furthermore, high- et al. 2008), providing the throughput required throughput protoplast transformation has for forward genetic screens. The random nature been used to perform quantitative characteriza- of this transformation method has been used to tion of large libraries of genetic elements in mine the underlying biology of plants by mu- Arabidopsis thaliana (Arabidopsis) and Sorghum tational T-DNA insertion as well as gene trap bicolor (sorghum) (Schaumberg et al. 2016). methods (reviewed in Springer 2000). Enhancer Precise methods for genomic inspection traps have been used to indicate the presence of and reverse genetics screens have also been im- nearby endogenous enhancer elements driving plemented in plants: for instance, CRISPR-Cas9 an orthogonal transcriptional activator such as genome editing has been applied in a number of GAL4-VP16. T-DNA insertion in the vicinity of plant model systems including Arabidopsis, Ni- an enhancer allows temporal and/or tissue-spe- cotiana spp., Solanum lycopersicum (tomato), cific expression of the activator, leading to re- Oryza sativa (rice), Triticum aestivum (wheat), stricted reporter gene expression according to Zea mays (maize), Citrus sinensis (orange), and 2 Cite this article as Cold Spring Harb Perspect Biol 2017;9:a023887 Downloaded from http://cshperspectives.cshlp.org/ on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press Synthetic Botany Marchantia polymorpha (Marchantia), at vary- dard parts for each new assembly (Ellis et al. ing efficiencies (reviewed in Bortesi and Fis- 2009; Liu et al. 2013; Patron 2014) unless stan- cher 2015). Notably, CRISPR-Cas9 genome dardized overlaps are used (Torella et al. 2013; editing has been successfully used to promote Casini et al. 2014). The application of Type IIS homologous recombination and to simultane- restriction enzymes for assembling standard ously target multiple loci in Arabidopsis and parts, known widely as “Golden Gate Cloning,” Nicotiana (Li et al. 2014), as well as to induce has become widely used as an alternative ap- targeted deletions in Nicotiana and rice (Low- proach because parts can be exchanged and as- der et al. 2015). Such methods are enabling sembled cheaply, easily, and in an automatable unprecedented efficiency of construct delivery, way without proprietary tools and reagents genome refactoring, and chassis engineering in (Engler et al. 2009; Sarrion-Perdigones et al. plants. 2011; Werner et al. 2012). Many commonly used sequences have been adapted for Type IIS assembly by various plant research laboratories A COMMON SYNTAX FOR SYNTHETIC (Sarrion-Perdigones et al. 2011; Weber et al. PLANT GENES 2011; Emami et al. 2013; Lampropoulos et al. Increasingly, synthetic biologists rely on the use 2013; Binder et al. 2014; Engler et al. 2014; Va- of modular DNA components to implement faee et al. 2014), and a common syntax to enable genetic circuits, along with