Plant synthetic biology: exploring the frontiers of sustainable agriculture and fundamental plant biology

Abstract

Plant synthetic biology allows for the reprogramming of biological systems to promote sustainable agriculture. It has a range of applications, including improving nutritional quality, engineering resilient crops, creating new materials, and producing various bioproducts. In addition, plant synthetic biology provides a new way to answer fundamental questions in plant biology. This Special Issue presents a collection of review articles that delve into the current and future state of plant synthetic biology. By designing and building genetic systems using standardized and interchangeable parts, synthetic biology is opening up possibilities to ‘reprogramme’ biological systems for improved sustainability. Plant synthetic biology offers even greater potential benefits to the bioeconomy, with the ability to engineer plants for improved nutritional value, reduced harmful compounds, and sustainable agriculture. At the same time, synthetic biology can help answer fundamental questions in plant biology by providing a means to systematically manipulate biological systems, giving biologists the possibility to test specific hypotheses about how these systems work and gain a deeper understanding of their underlying mechanisms. Despite the potential benefits, we are still limited by our knowledge of plant systems. One of the biggest challenges is to fully understand the complex interactions of multicellular organisms occurring at the molecular, cellular, and environmental levels. For instance, one of the big questions in plant biology is: how are cells able to handle random variations in gene expression, maintain stability, and at the same time adapt to changes in the environment? The basis for this Special Issue was the lively discussions that took place during the ‘Plant Synthetic Biology’ conference held in Barcelona, Spain, in 2022. The conference brought together researchers from various fields, including those working on enabling molecular tools, green microorganisms and new plant model systems, social dimensions of synthetic biology, synthetic metabolism for agricultural and industrial applications, synthetic genomics, modelling, bioinformatics, automation, and synthetic signalling and reconstruction biology in orthogonal systems. This Special Issue delves into some of these topics, with reviews exploring the challenges and potential applications of synthetic biology in plant biology. As the response to environmental variables is critical to plants, plant synthetic biology relies on the design of synthetic gene circuits (SGCs) capable of recognizing external inputs and processing them to generate a desired output. These circuits are made from standardized, modular, and well-characterized building parts in the form of enhancers, untranslated elements, spacers, terminators, and coding sequences (Kocaoglan et al., 2023). The generation of these transcriptional units has greatly benefited from the application of Type IIS restriction enzymes for DNA assembly technologies (Weber et al., 2011; Engler et al., 2014; Kocaoglan et al., 2023). In this regard, researchers in plant biology have proposed a common syntax that enables the exchange of DNA parts for plant synthetic biology (Patron et al., 2015). Different transcriptional units are then assembled into higher order functional modules that perform specific tasks. In fact, a classification and a common syntax to refer to these modules are still lagging in comparison with those of DNA parts. In this Special Issue, Vazquez-Vilar et al. (2023) approach this by offering a categorization of functional modules based on their role as sensors, processors, and actuators. This abstraction allows us to conceptualize complex biological systems as modular components with defined inputs and outputs, thereby simplifying the design and construction of new systems. The review by Vazquez-Vilar et al. (2023) covers synthetic transcriptional gene circuits, tools and technologies for gene circuit design, and recent advances in SGC design, including key components such as programmable transcriptional regulators, synthetic promoters, and Boolean logic gates. It also highlights current challenges and future directions for assembling functional circuits in transgenic plants, emphasizing the need for continued advancements in areas such as computer-assisted design and testing methodologies. The idea of gene targeting which involves the precise modification of a genome to introduce, alter, or eliminate specific traits, is a stepping stone towards plant synthetic biology. While it was shown that gene targeting could enable genome modifications through homologous recombination (Paszkowski et al., 1988), the error-prone nature of the non-homologous end joining (NHEJ) DNA repair mechanism that is predominantly used in higher plants significantly affects its efficiency (Puchta, 2005). The advent of genome editing technologies capable of inducing double-strand breaks in desired loci has revitalized the concept of gene targeting (Huang and Puchta, 2019; Guzmán-Benito et al., 2023). Despite the use of CRISPR/Cas [clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein] technologies to target specific loci, the dominance of NHEJ as a double-strand break repair mechanism in plants is an important bottleneck for crop engineering. Guzmán-Benito et al. (2023) discuss alternative approaches towards bypassing the NHEJ pathway and, at the same time, the need to fully understand the nature of homologous recombination to achieve a fast and efficient gene targeting through the use of CRISPR/Cas technologies. Particular attention is given to shifting the balance of DNA repair to other mechanisms based on homologous recombination (HR) in which a homologous template strand is used to repair the breaks, giving high fidelity to the repair. On the other hand, new research has emerged towards improving CRISPR/Cas for a better gene targeting (also discussed in Guzmán-Benito et al., 2023) Before CRISPR/Cas, the manipulation of gene expression relied mainly on the use of small RNAs (sRNAs), which are essential regulators of gene expression in all eukaryotes. Conversely, the integration of transgenes is often compromised by these mechanisms of gene silencing. It is common practice to co-express suppressors of RNA silencing with the target genes to be introduced. Guzmán-Benito et al. (2023) reflect on innovative approaches that incorporate sRNA technologies and CRISPR/Cas for the purpose of improving gene targeting; for instance, the use of artificial miRNAs targeting single guide RNAs (sgRNAs) to act as ‘sponges’ to block silencing. It is clear that the addition of auxiliary components for a more efficient gene targeting implies expanding the current toolset to generate synthetic genetic circuits. While the creation of these circuits has benefited from the establishment of molecular cloning techniques and the reduction of costs in gene synthesis (as reviewed by Vazquez-Vilar et al., 2023), the addition of new parts in the form of enhancers, terminators, and spacers for testing complex arrangements of CRISPR/Cas elements is necessary. Milito et al. (2023) discuss the use of Chlamydomonas as a chassis for high-throughput enhancer characterization. In this regard, the selection of the chassis becomes fundamental in the design–build–test cycle. It is clear that a single-cell system that is easily transformable can help screen for successful gene targeting events via CRISPR/Cas. Protoplasts offer an interesting alternative for this. New work on the use of protoplasts for gene editing and the challenges associated with identifying regenerants that possess the desired edits is well explored in this issue by Guzmán-Benito et al. (2023) and Reyna-Llorens et al. (2023). Protoplasts have been at the forefront of plant research for over a century, providing a valuable tool for the study of plant physiology, cell ultrastructure, and genetics (Yoo et al., 2007). However, recent advances in synthetic biology have propelled protoplasts into the spotlight as a tool to accelerate the ‘design–build–test–learn’ cycle of genetic engineering in plants. Protoplast isolation and transfection have enabled high-throughput characterization of genetic parts, allowing for the control of gene expression in synthetic genetic circuits. While protoplast regeneration remains a challenging process, significant progress has been made in understanding the molecular basis of somatic embryogenesis, stress-induced alterations in gene expression, and the overexpression of master regulators. This progress could lead to the development of a powerful tool in plant breeding and genetic engineering. Reyna-Llorens et al. (2023) highlight the potential of combining protoplast assays with synthetic biology techniques to create high-throughput discovery platforms for studying gene function, plant metabolic diversity, and the development of novel breeding and synthetic biology tools. Another chassis with a lot of potential for synthetic biology is the unicellular algae Chlamydomonas reinhardtii. This green alga has gained increasing attention as a suitable host for producing high-value chemicals and recombinant proteins (Scaife et al., 2015). Recently granted GRAS (Generally Recognized As Safe) status, this organism, with its expanding genetic toolkit, holds significant promise as a safe and sustainable source of high-value compounds, serving as a carbon-fixing biofactory. In the past, Chlamydomonas has shown poor expression and instability of foreign genes, often losing transgene expression over time. Recent developments involving the UVM4/11 strains and a mutant strain obtained from the Chlamydomonas Library Project (CLiP) have helped to overcome these instability issues to some extent (Neupert et al., 2020). Bioengineering microalgal strains to optimize and modify their metabolic outputs will be critical. This will involve achieving precise and tuneable control over transgene expression, necessitating the development and rational design of synthetic promoters. However, the current repertoire of functional synthetic promoters for Chlamydomonas, and microalgae generally, is limited compared with other commercial hosts. This emphasizes the need to expand the gene expression toolbox for microalgae. Milito et al. (2023) discuss strategies for expanding this toolset, focusing on the need to advance in the development of synthetic promoters for Chlamydomonas. They draw on high-throughput studies performed in other model systems that could be applicable to microalgae and propose novel approaches to interrogate algal promoters. Furthermore, they outline the significant limitations hindering microalgal promoter development and provide novel suggestions and perspectives on overcoming these challenges. The miniaturization of controlled environments for manipulating and screening cells has shown great promise in plant biology, as exemplified by the creation of small chips that enable single protoplasts to traverse a channel integrated with a screening system. This breakthrough opens up opportunities to develop high-throughput platforms for testing genetic circuits and studying various aspects of plant growth and development [discussed in Reyna-Llorens et al. (2023) and Kaiser et al. (2023) in this Special Issue]. However, a ‘common syntax’ for microfluidics, akin to that used for genetic circuits, could facilitate the seamless integration of independently developed chips, leading to efficient selection and regeneration of cells with desired phenotypes. Furthermore, microdevices can be used to study whole tissues and individuals, and one area of research where this approach has shown promise is root biology. Roots constantly sense and process signals that trigger various developmental responses, making it necessary to control many of the variables that roots would encounter in nature to study them effectively. Kaiser et al. (2023) present a compelling overview of the utilization of synthetic environments for observing, manipulating, and analysing roots. These setups encompass various components, such as co-cultivation with microbes, the introduction of physical obstacles, and the generation of heterogeneous microenvironments, effectively replicating the behaviours exhibited by soil communities. Moreover, the review by Kaiser et al. (2023) serves as an excellent resource for researchers seeking to integrate these technologies into their investigations of the intricate network behaviour displayed by soil communities, offering a valuable starting point for further exploration in this field. Optimized function requires optimized form. For instance, a plant with more branches could bear more fruits or grains, leading to increased yield (Guo et al., 2020; Zheng et al., 2020). Similarly, growing larger crops can also enhance harvest output (Si et al., 2016). Consequently, rational reprogramming of plant growth and development could be a game-changer for the next Green Revolution. The intersection of synthetic and plant developmental biology offers promising strategies for designing new plant forms (Ebrahimkhani and Ebisuya, 2019; Zarkesh et al., 2022). Given the complexities and unpredictabilities of genetically engineering plant development, it is crucial to adopt a precise and systematic approach. In this context, Gediz Kocaoglan and colleagues have provided an insightful overview of the latest synthetic biology technologies that can be utilized for modifying plant development (Kocaoglan et al., 2023). Furthermore, they illustrated three examples of how these synthetic biology tools have been applied to engineer plant development for shoot architecture (Khakhar et al., 2018), the reproductive system (Ochoa-Fernandez et al., 2020), and root architecture (Brophy et al., 2022). Through the integration of synthetic biology principles and tools, plant scientists can benefit from and accelerate their discoveries and applications to more sustainable agriculture. However, the challenge of incorporating synthetic biology to improve the nutritional quality of food, enhance plant resistance to biotic and abiotic stressors, or produce valuable compounds lies in the complexity of plant systems and our limited understanding of their intricate biological processes. The latest advances in synthetic biology could offer a systematic approach to understanding these processes and overcoming the challenges. We would like to thank all the attendees and speakers who made the Plant Synthetic Biology Conference in Barcelona a success. Your contributions and active participation paved the way for the creation of this Special Issue. We also thank Dr Robertas Ursache for his insightful comments on an earlier version of this editorial. The authors declare no conflict of interest. We gratefully acknowledge the financial support from the Ministry of Science and Innovation and Innovation-State Research Agency (AEI), through the accreditation Center of Excellence “Severo Ochoa” 2019, CEX2019-000902-S funded by MCIN/AEI/ 10.13039/501100011033, and grant RYC2020-028880-I for JS Y funded by MCIN/AEI/ 10.13039/501100011033 and by “European Union NextGenerationEU/PRTR”.

Topics & Concepts