Engineered plants provide a photosynthetic platform for the production of diverse human milk oligosaccharides.

Human milk oligosaccharides (HMOs) are a diverse class of carbohydrates which support the health and development of infants. The vast health benefits of HMOs have made them a commercial target for microbial production; however, producing the approximately 200 structurally diverse HMOs at scale has proved difficult. Here we produce a diversity of HMOs by leveraging the robust carbohydrate anabolism of plants. This diversity includes high-value and complex HMOs, such as lacto-N-fucopentaose I. HMOs produced in transgenic plants provided strong bifidogenic properties, indicating their ability to serve as a prebiotic supplement with potential applications in adult and infant health. Technoeconomic analyses demonstrate that producing HMOs in plants provides a path to the large-scale production of specific HMOs at lower prices than microbial production platforms. Our work demonstrates the promise in leveraging plants for the low-cost and sustainable production of HMOs.

Systematic identification of transcriptional activation domains from non-transcription factor proteins in plants and yeast.

Transcription factors can promote gene expression through activation domains. Whole-genome screens have systematically mapped activation domains in transcription factors but not in non-transcription factor proteins (e.g., chromatin regulators and coactivators). To fill this knowledge gap, we employed the activation domain predictor PADDLE to analyze the proteomes of Arabidopsis thaliana and Saccharomyces cerevisiae. We screened 18,000 predicted activation domains from >800 non-transcription factor genes in both species, confirming that 89% of candidate proteins contain active fragments. Our work enables the annotation of hundreds of nuclear proteins as putative coactivators, many of which have never been ascribed any function in plants. Analysis of peptide sequence compositions reveals how the distribution of key amino acids dictates activity. Finally, we validated short, "universal" activation domains with comparable performance to state-of-the-art activation domains used for genome engineering. Our approach enables the genome-wide discovery and annotation of activation domains that can function across diverse eukaryotes.

Engineering Brassica Crops to Optimize Delivery of Bioactive Products Postcooking.

Glucosinolates are plant-specialized metabolites that can be hydrolyzed by glycosyl hydrolases, called myrosinases, creating a variety of hydrolysis products that benefit human health. While cruciferous vegetables are a rich source of glucosinolates, they are often cooked before consumption, limiting the conversion of glucosinolates to hydrolysis products due to the denaturation of myrosinases. Here we screen a panel of glycosyl hydrolases for high thermostability and engineer the Brassica crop, broccoli (Brassica oleracea L.), for the improved conversion of glucosinolates to chemopreventive hydrolysis products. Our transgenic broccoli lines enabled glucosinolate hydrolysis to occur at higher cooking temperatures, 20 °C higher than in wild-type broccoli. The process of cooking fundamentally transforms the bioavailability of many health-relevant bioactive compounds in our diet. Our findings demonstrate the promise of leveraging genetic engineering to tailor crops with novel traits that cannot be achieved through conventional breeding and improve the nutritional properties of the plants we consume.

Cynipid wasps systematically reprogram host metabolism and restructure cell walls in developing galls.

Many insects have evolved the ability to manipulate plant growth to generate extraordinary structures called galls, in which insect larva can develop while being sheltered and feeding on the plant. In particular, cynipid (Hymenoptera: Cynipidae) wasps have evolved to form morphologically complex galls and generate an astonishing array of gall shapes, colors, and sizes. However, the biochemical basis underlying these remarkable cellular and developmental transformations remains poorly understood. A key determinant in plant cellular development is cell wall deposition that dictates the physical form and physiological function of newly developing cells, tissues, and organs. However, it is unclear to what degree cell walls are restructured to initiate and support the formation of new gall tissue. Here, we characterize the molecular alterations underlying gall development using a combination of metabolomic, histological, and biochemical techniques to elucidate how valley oak (Quercus lobata) leaf cells are reprogrammed to form galls. Strikingly, gall development involves an exceptionally coordinated spatial deposition of lignin and xylan to form de novo gall vasculature. Our results highlight how cynipid wasps can radically change the metabolite profile and restructure the cell wall to enable the formation of galls, providing insights into the mechanism of gall induction and the extent to which plants can be entirely reprogrammed to form unique structures and organs.

Systematic identification of transcriptional activator domains from non-transcription factor proteins in plants and yeast.

Transcription factors promote gene expression via trans-regulatory activation domains. Although whole genome scale screens in model organisms (e.g. human, yeast, fly) have helped identify activation domains from transcription factors, such screens have been less extensively used to explore the occurrence of activation domains in non-transcription factor proteins, such as transcriptional coactivators, chromatin regulators and some cytosolic proteins, leaving a blind spot on what role activation domains in these proteins could play in regulating transcription. We utilized the activation domain predictor PADDLE to mine the entire proteomes of two model eukaryotes, Arabidopsis thaliana and Saccharomyces cerevisiae ( 1 ). We characterized 18,000 fragments covering predicted activation domains from >800 non-transcription factor genes in both species, and experimentally validated that 89% of proteins contained fragments capable of activating transcription in yeast. Peptides with similar sequence composition show a broad range of activities, which is explained by the arrangement of key amino acids. We also annotated hundreds of nuclear proteins with activation domains as putative coactivators; many of which have never been ascribed any function in plants. Furthermore, our library contains >250 non-nuclear proteins containing peptides with activation domain function across both eukaryotic lineages, suggesting that there are unknown biological roles of these peptides beyond transcription. Finally, we identify and validate short, 'universal' eukaryotic activation domains that activate transcription in both yeast and plants with comparable or stronger performance to state-of-the-art activation domains. Overall, our dual host screen provides a blueprint on how to systematically discover novel genetic parts for synthetic biology that function across a wide diversity of eukaryotes. Significance Statement: Activation domains promote transcription and play a critical role in regulating gene expression. Although the mapping of activation domains from transcription factors has been carried out in previous genome-wide screens, their occurrence in non-transcription factors has been less explored. We utilize an activation domain predictor to mine the entire proteomes of Arabidopsis thaliana and Saccharomyces cerevisiae for new activation domains on non-transcription factor proteins. We validate peptides derived from >750 non-transcription factor proteins capable of activating transcription, discovering many potentially new coactivators in plants. Importantly, we identify novel genetic parts that can function across both species, representing unique synthetic biology tools.

The trans-regulatory landscape of gene networks in plants.

The transcriptional effector domains of transcription factors play a key role in controlling gene expression; however, their functional nature is poorly understood, hampering our ability to explore this fundamental dimension of gene regulatory networks. To map the trans-regulatory landscape in a complex eukaryote, we systematically characterized the putative transcriptional effector domains of over 400 Arabidopsis thaliana transcription factors for their capacity to modulate transcription. We demonstrate that transcriptional effector activity can be integrated into gene regulatory networks capable of elucidating the functional dynamics underlying gene expression patterns. We further show how our characterized domains can enhance genome engineering efforts and reveal how plant transcriptional activators share regulatory features conserved across distantly related eukaryotes. Our results provide a framework to systematically characterize the regulatory role of transcription factors at a genome-scale in order to understand the transcriptional wiring of biological systems.

Plant-based engineering for production of high-valued natural products.

Covering: up to March 2022Plants are a unique source of complex specialized metabolites, many of which play significant roles in human society. In many cases, however, the availability of these metabolites from naturally occurring sources fails to meet current demands. Thus, there is much interest in expanding the production capacity of target plant molecules. Traditionally, plant breeding, chemical synthesis, and microbial fermentation are considered the primary routes towards large scale production of natural products. Here, we explore the advances, challenges, and future of plant engineering as a complementary path. Although plants are an integral part of our food and agricultural systems and sustain an extensive array of chemical constituents, their complex genetics and physiology have prevented the optimal exploitation of plants as a production chassis. We highlight emerging engineering tools and scientific advances developed in recent years that have improved the prospects of using plants as a sustainable and scalable production platform. We also discuss technological limitations and overall economic outlook of plant-based production of natural products.

From breeding to genome design: A genomic makeover for potatoes.

Potato breeding efforts have long been hindered by the genetic consequences of millennia of clonal propagation. To mitigate genomic constraints, Zhang et al. leverage an unprecedented scale of sequencing and marker-assisted breeding to unlock traits that have not been possible through classical breeding, providing a blueprint for plant genome design.

Construction of DNA Tools for Hyperexpression in Marchantia Chloroplasts.

Chloroplasts are attractive platforms for synthetic biology applications since they are capable of driving very high levels of transgene expression, if mRNA production and stability are properly regulated. However, plastid transformation is a slow process and currently limited to a few plant species. The liverwort Marchantia polymorpha is a simple model plant that allows rapid transformation studies; however, its potential for protein hyperexpression has not been fully exploited. This is partially due to the fact that chloroplast post-transcriptional regulation is poorly characterized in this plant. We have mapped patterns of transcription in Marchantia chloroplasts. Furthermore, we have obtained and compared sequences from 51 bryophyte species and identified putative sites for pentatricopeptide repeat protein binding that are thought to play important roles in mRNA stabilization. Candidate binding sites were tested for their ability to confer high levels of reporter gene expression in Marchantia chloroplasts, and levels of protein production and effects on growth were measured in homoplastic transformed plants. We have produced novel DNA tools for protein hyperexpression in this facile plant system that is a test-bed for chloroplast engineering.

CRISPR-Act3.0 for highly efficient multiplexed gene activation in plants.

RNA-guided CRISPR activation (CRISPRa) systems have been developed in plants. However, the simultaneous activation of multiple genes remains challenging. Here, we develop a highly robust CRISPRa system working in rice, Arabidopsis and tomato, CRISPR-Act3.0, through systematically exploring different effector recruitment strategies and various transcription activators based on deactivated Streptococcus pyogenes Cas9 (dSpCas9). The CRISPR-Act3.0 system results in fourfold to sixfold higher activation than the state-of-the-art CRISPRa systems. We further develop a tRNA-gR2.0 (single guide RNA 2.0) expression system enabling CRISPR-Act3.0-based robust activation of up to seven genes for metabolic engineering in rice. In addition, CRISPR-Act3.0 allows the simultaneous modification of multiple traits in Arabidopsis, which are stably transmitted to the T3 generations. On the basis of CRISPR-Act3.0, we elucidate guide RNA targeting rules for effective transcriptional activation. To target T-rich protospacer adjacent motifs (PAMs), we transfer this activation strategy to CRISPR-dCas12b and further improve the dAaCas12b-based CRISPRa system. Moreover, we develop a potent near-PAM-less CRISPR-Act3.0 system on the basis of the SpRY dCas9 variant, which outperforms the dCas9-NG system in both activation potency and targeting scope. Altogether, our study has substantially improved the CRISPRa technology in plants and provided plant researchers a powerful toolbox for efficient gene activation in foundational and translational research.

Rapid and Modular DNA Assembly for Transformation of Marchantia Chloroplasts.

The bryophyte Marchantia polymorpha , has attracted significant attention as a powerful experimental system for studying aspects of plant biology including synthetic biology applications. We describe an efficient and simple recursive Type IIS DNA assembly method for the generation of DNA constructs for chloroplast genome manipulation, and an optimized technique for Marchantia chloroplast genome transformation. The utility of the system was demonstrated by the expression of a chloroplast codon-optimized cyan fluorescent protein.

Response to comment on 'Lack of evidence for associative learning in pea plants'.

In 2016 Gagliano et al. reported evidence for associative learning in plants (Gagliano et al., 2016). A subsequent attempt to replicate this finding by the present author was not successful (Markel, 2020). Gagliano et al. attribute this lack of replication to differences in the experimental set-ups used in the original work and the replication attempt (Gagliano et al., 2020). Here, based on a comparison of the two set-ups, I argue that these differences are unable to explain the lack of replication in Markel, 2020.

Lack of evidence for associative learning in pea plants.

Gagliano et al. (Learning by association in plants, 2016) reported associative learning in pea plants. Associative learning has long been considered a behavior performed only by animals, making this claim particularly newsworthy and interesting. In the experiment, plants were trained in Y-shaped mazes for 3 days with fans and lights attached at the top of the maze. Training consisted of wind consistently preceding light from either the same or the opposite arm of the maze. When plant growth forced a decision between the two arms of the maze, fans alone were able to influence growth direction, whereas the growth direction of untrained plants was not affected by fans. However, a replication of their protocol failed to demonstrate the same result, calling for further verification and study before mainstream acceptance of this paradigm-shifting phenomenon. This replication attempt used a larger sample size and fully blinded analysis.

Associative learning is a simple learning ability found in most animals, which involves linking together two different cues. For example, the dogs in Pavlov's famous experiment were trained to associate sound with the arrival of food, and eventually started salivating upon hearing the sound alone. Plants, like animals, are capable of complex behaviors. The snapping leaves of a Venus fly trap or the sun-tracking abilities of sunflowers are examples of instinctive responses to environmental cues that have evolved over many generations. Whether or not plants can learn during their lifetimes has remained unknown. A handful of studies have tested for associative learning in plants, the most convincing of which was published in 2016. In this study, pea plants were exposed to two signals: light, the plant version of dog food, and wind, equivalent to the sound in Pavlov's experiment. Just as dogs salivate in response to food, plants instinctively grow towards light, whereas air flow does not affect the direction of growth. The plants were grown inside Y-shaped mazes and their 'selection' of one particular arm was used as a 'read-out' of learned behavior. The experiments trained growing plants by exposing them to wind and light from either the same direction or opposite directions. Once the plants were at the point of 'choosing' between the two arms, they were exposed to wind in the absence of light. Wind by itself appeared to influence the direction the trained plants took, with wind attracting plants trained with wind and light together and repelling plants trained with wind and light apart. Untrained plants remained unaffected, making random selections. These observations were interpreted as the strongest evidence of associative learning in plants and if true would have great scientific and philosophical significance. Kasey Markel therefore set out to confirm and expand on these findings by replicating the 2016 study. As many conditions as possible were kept identical, such as the training regime. The new experiments also used more plants and, most importantly, were done 'blind' meaning the people recording the data did not know how the plants had been trained. This ensured the expectations of the researcher would not influence the final results. The new study found no evidence for associative learning, but did not rule it out altogether. This is because some experimental details in the first study remained unknown, such as the exact model of lights and fans originally used. This work demonstrates the importance of replicating scientific experiments. In the future, Markel hopes their results will pave the way for further, rigorous testing of the hypothesis that plants can learn.

Systematic Tools for Reprogramming Plant Gene Expression in a Simple Model, Marchantia polymorpha.

We present the OpenPlant toolkit, a set of interlinked resources and techniques to develop Marchantia as testbed for bioengineering in plants. Marchantia is a liverwort, a simple plant with an open form of development that allows direct visualization of gene expression and dynamics of cellular growth in living tissues. We describe new techniques for simple and efficient axenic propagation and maintenance of Marchantia lines with no requirement for glasshouse facilities. Marchantia plants spontaneously produce clonal propagules within a few weeks of regeneration, and lines can be amplified million-fold in a single generation by induction of the sexual phase of growth, crossing, and harvesting of progeny spores. The plant has a simple morphology and genome with reduced gene redundancy, and the dominant phase of its life cycle is haploid, making genetic analysis easier. We have built robust Loop assembly vector systems for nuclear and chloroplast transformation and genome editing. These have provided the basis for building and testing a modular library of standardized DNA elements with highly desirable properties. We have screened transcriptomic data to identify a range of candidate genes, extracted putative promoter sequences, and tested them in vivo to identify new constitutive promoter elements. The resources have been combined into a toolkit for plant bioengineering that is accessible for laboratories without access to traditional facilities for plant biology research. The toolkit is being made available under the terms of the OpenMTA and will facilitate the establishment of common standards and the use of this simple plant as testbed for synthetic biology.

Plant Biosystems Design Research Roadmap 1.0.

Human life intimately depends on plants for food, biomaterials, health, energy, and a sustainable environment. Various plants have been genetically improved mostly through breeding, along with limited modification via genetic engineering, yet they are still not able to meet the ever-increasing needs, in terms of both quantity and quality, resulting from the rapid increase in world population and expected standards of living. A step change that may address these challenges would be to expand the potential of plants using biosystems design approaches. This represents a shift in plant science research from relatively simple trial-and-error approaches to innovative strategies based on predictive models of biological systems. Plant biosystems design seeks to accelerate plant genetic improvement using genome editing and genetic circuit engineering or create novel plant systems through de novo synthesis of plant genomes. From this perspective, we present a comprehensive roadmap of plant biosystems design covering theories, principles, and technical methods, along with potential applications in basic and applied plant biology research. We highlight current challenges, future opportunities, and research priorities, along with a framework for international collaboration, towards rapid advancement of this emerging interdisciplinary area of research. Finally, we discuss the importance of social responsibility in utilizing plant biosystems design and suggest strategies for improving public perception, trust, and acceptance.

Biosystems Design to Accelerate C3-to-CAM Progression.

Global demand for food and bioenergy production has increased rapidly, while the area of arable land has been declining for decades due to damage caused by erosion, pollution, sea level rise, urban development, soil salinization, and water scarcity driven by global climate change. In order to overcome this conflict, there is an urgent need to adapt conventional agriculture to water-limited and hotter conditions with plant crop systems that display higher water-use efficiency (WUE). Crassulacean acid metabolism (CAM) species have substantially higher WUE than species performing C3 or C4 photosynthesis. CAM plants are derived from C3 photosynthesis ancestors. However, it is extremely unlikely that the C3 or C4 crop plants would evolve rapidly into CAM photosynthesis without human intervention. Currently, there is growing interest in improving WUE through transferring CAM into C3 crops. However, engineering a major metabolic plant pathway, like CAM, is challenging and requires a comprehensive deep understanding of the enzymatic reactions and regulatory networks in both C3 and CAM photosynthesis, as well as overcoming physiometabolic limitations such as diurnal stomatal regulation. Recent advances in CAM evolutionary genomics research, genome editing, and synthetic biology have increased the likelihood of successful acceleration of C3-to-CAM progression. Here, we first summarize the systems biology-level understanding of the molecular processes in the CAM pathway. Then, we review the principles of CAM engineering in an evolutionary context. Lastly, we discuss the technical approaches to accelerate the C3-to-CAM transition in plants using synthetic biology toolboxes.

Defining and engineering bioenergy plant feedstock ideotypes.

Ideotypes are theoretical archetypes of crops which serve as a practical framework for plant breeders to critically evaluate what traits they should be targeting for specific applications. With advances in plant biotechnology and a growing urgency to adopt more sustainable practices across our economy, new uses for crops as bioenergy feedstocks may pivot our definition of an ideal crop that is engineered for biomass and bioenergy production, in contrast to food production. Although there is a plethora of specific applications to which plant engineering efforts can contribute, here we highlight recent advances in two broad areas of research: increasing available plant biomass and engineering production of higher value co-products.

Cryptic Amyloidogenic Elements in the 3' UTRs of Neurofilament Genes Trigger Axonal Neuropathy.

Abnormal protein aggregation is observed in an expanding number of neurodegenerative diseases. Here, we describe a mechanism for intracellular toxic protein aggregation induced by an unusual mutation event in families affected by axonal neuropathy. These families carry distinct frameshift variants in NEFH (neurofilament heavy), leading to a loss of the terminating codon and translation of the 3' UTR into an extra 40 amino acids. In silico aggregation prediction suggested the terminal 20 residues of the altered NEFH to be amyloidogenic, which we confirmed experimentally by serial deletion analysis. The presence of this amyloidogenic motif fused to NEFH caused prominent and toxic protein aggregates in transfected cells and disrupted motor neurons in zebrafish. We identified a similar aggregation-inducing mechanism in NEFL (neurofilament light) and FUS (fused in sarcoma), in which mutations are known to cause aggregation in Charcot-Marie-Tooth disease and amyotrophic lateral sclerosis, respectively. In summary, we present a protein-aggregation-triggering mechanism that should be taken into consideration during the evaluation of stop-loss variants.