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Nanomaterials have emerged as an invaluable tool for the delivery of biomolecules such as DNA and RNA, with various applications in genetic engineering and post-transcriptional genetic manipulation. Alongside this development, there has been an increasing use of polymer-based techniques, such as polyethylenimine (PEI), to electrostatically load polynucleotide cargoes onto nanomaterial carriers. However, there remains a need to assess nanomaterial properties, conjugation conditions, and biocompatibility of these nanomaterial-polymer constructs, particularly for use in plant systems. In this work, we develop mechanisms to optimize DNA loading on single-walled carbon nanotubes (SWNTs) with a library of polymer-SWNT constructs and assess DNA loading ability, polydispersity, and both chemical and colloidal stability. Counterintuitively, we demonstrate that polymer hydrolysis from nanomaterial surfaces can occur depending on polymer properties and attachment chemistries, and we describe mitigation strategies against construct degradation. Given the growing interest in delivery applications in plant systems, we also assess the stress response of plants to polymer-based nanomaterials and provide recommendations for future design of nanomaterial-based polynucleotide delivery strategies.

The global SARS-CoV-2 coronavirus pandemic has led to a surging demand for rapid and efficient viral infection diagnostic tests, generating a supply shortage in diagnostic test consumables including nucleic acid extraction kits. Here, we develop a modular method for high-yield extraction of viral single-stranded nucleic acids by using “capture” ssDNA sequences attached to carbon nanotubes. Target SARS-CoV-2 viral RNA can be captured by ssDNA-nanotube constructs via hybridization and separated from the liquid phase in a single-tube system with minimal chemical reagents, for downstream quantitative reverse transcription polymerase chain reaction (RT-qPCR) detection. This nanotube-based extraction method enables 100% extraction yield of target SARS-CoV-2 RNA from phosphate-buffered saline in comparison to ∼20% extraction yield when using a commercial silica-column kit. Notably, carbon nanotubes enable extraction of nucleic acids directly from 50% human saliva with a similar efficiency as achieved with commercial DNA/RNA extraction kits, thereby bypassing the need for further biofluid purification and avoiding the use of commercial extraction kits. Carbon nanotube-based extraction of viral nucleic acids facilitates high-yield and high-sensitivity identification of viral nucleic acids such as the SARS-CoV-2 viral genome with a reduced reliance on reagents affected by supply chain obstacles.

Protein delivery to plants offers many opportunities for plant bioengineering via gene editing and through direction of protein-protein interactions. However, the delivery and confirmation of successful protein delivery to plants presents both practical and analytical challenges. We present a GFP bimolecular fluorescence complementation-based tool, delivered complementationin planta(DCIP), which allows for unambiguous and quantitative measurement of protein delivery in leaves. Using DCIP, we demonstrate cell-penetrating peptide mediated cytosolic delivery of peptides and recombinant proteins inNicotiana benthamiana. We show that DCIP enables measurement of delivery efficiency and enables functional screening of cell penetrating peptide efficacies for in-plant protein delivery. Finally, we demonstrate that DCIP detects cell penetrating peptide mediated delivery of recombinantly expressed proteins such as mCherry and Lifeact into intact leaves. Finally, we also demonstrate, for the first time, delivery of a recombinant plant transcription factor, WUSCHEL (AtWUS), inN. benthamiana. RT-qPCR analysis of AtWUS delivery inArabidopsisseedlings also suggests delivered WUS can recapitulate AtWUS-overexpression transcriptional changes. All combined, DCIP offers a new and powerful tool for interrogating cytosolic delivery of proteins in plants and highlights future avenues for engineering plant physiology.

Targeted downregulation of select endogenous plant genes is known to confer disease or pest resistance in crops and is routinely accomplished via transgenic modification of plants for constitutive gene silencing. An attractive alternative to the use of transgenics or pesticides in agriculture is the use of a ‘green’ alternative known as RNAi, which involves the delivery of siRNAs that downregulate endogenous genes to confer resistance. However, siRNA is a molecule that is highly susceptible to enzymatic degradation and is difficult to deliver across the lignin-rich and multi-layered plant cell wall that poses the dominant physical barrier to biomolecule delivery in plants. We have demonstrated that DNA nanostructures can be utilized as a cargo carrier for direct siRNA delivery and gene silencing in mature plants. The size, shape, compactness and stiffness of the DNA nanostructure affect both internalization into plant cells and subsequent gene silencing efficiency. Herein, we provide a detailed protocol that can be readily adopted with standard biology benchtop equipment to generate geometrically optimized DNA nanostructures for transgene-free and force-independent siRNA delivery and gene silencing in mature plants. We further discuss how such DNA nanostructures can be rationally designed to efficiently enter plant cells and deliver cargoes to mature plants, and provide guidance for DNA nanostructure characterization, storage and use. The protocol described herein can be completed in 4 d. This protocol describes how to engineer DNA nanostructures with different sizes, shapes and mechanical properties; load them with a siRNA cargo; and evaluate their ability to silence genes in mature tobacco plants.

Gravitropism is a finely regulated tropistic response based on the plant perception of directional cues. Such perception allows them to direct shoot growth upwards, above ground, and root growth downwards, into the soil, anchoring the plant to acquire water and nutrients. Gravity sensing occurs in specialized cells and depends on auxin distribution, regulated by influx/efflux carriers. Here we report that AtHB40, encoding a transcription factor of the homeodomain-leucine zipper I family, was expressed in the columella and the root tip. Athb40 mutants exhibited longer primary roots. Enhanced primary root elongation was in agreement with a higher number of cells in the transition zone and the induction of CYCLINB transcript levels. Moreover, athb40 mutants and AtHB40 overexpressors displayed enhanced and delayed gravitropistic responses, respectively. These phenotypes were associated with altered auxin distribution and deregulated expression of the auxin transporters LAX2, LAX3, and PIN2. Accordingly, lax2 and lax3 mutants also showed an altered gravitropistic response, and LAX3 was identified as a direct target of AtHB40. Furthermore, AtHB40 is induced by AtHB53 when the latter is upregulated by auxin. Altogether, these results indicate that AtHB40 modulates cell division and auxin distribution in the root tip thus altering primary root length and gravitropism.

As sessile organisms, plants must adapt to a changing environment, sensing variations in resource availability and modifying their development in response. Light is one of the most important resources for plants, and its perception by sensory photoreceptors (e.g. phytochromes) and subsequent transduction into long-term transcriptional reprogramming have been well characterized. Chromatin changes have been shown to be involved in photomorphogenesis. However, the initial short-term transcriptional changes produced by light and what factors enable these rapid changes are not well studied. Here, we identify rapidly light-responsive, PIF (Phytochrome Interacting Factor) direct-target genes (LRP-DTGs). We found that a majority of these genes also show rapid changes in Histone 3 Lysine-9 acetylation (H3K9ac) in response to the light signal. Detailed time-course analysis of transcriptional and chromatin changes showed that, for light-repressed genes, H3K9 deacetylation parallels light-triggered transcriptional repression, while for light-induced genes, H3K9 acetylation appeared to somewhat precede light-activated transcription. However, real-time imaging of transcription elongation revealed that, in fact, H3K9 acetylation also parallels transcriptional induction. Collectively, the data raise the possibility that light-induced transcriptional and chromatin-remodeling processes are mechanistically intertwined. Histone modifying proteins involved in long term light responses do not seem to have a role in this fast response, indicating that different factors might act at different stages of the light response. This work not only advances our understanding of plant responses to light, but also unveils a system in which rapid chromatin changes in reaction to an external signal can be studied under natural conditions.

Plant synthetic biology requires precise characterization of genetic elements to construct complex genetic circuits that can improve plant traits or confer them with new characteristics. Transcriptional reporter assays are essential to quantify the effect of gene expression regulator elements. Additionally, transcriptional reporter systems are a key tool in understanding control of gene expression in biology. In this work, we construct and characterize a dual color luciferase ratiometric reporter system that possesses several advantages over currently used reporters. It is ratiometric, thus reducing variability and increasing consistency between experiments; it is fast, as both reporters can be measured at the same time in a single reaction, and it is less expensive to perform than current dual luciferase reporter assays. We have validated our system quantifying the transcriptional capability of a panel of promoters and terminators commonly used in synthetic biology with a broad range of expression magnitudes, and in a biologically relevant system, nitrate response.

Exogenous biomolecule delivery into plants is difficult because the plant cell wall poses a dominant transport barrier, thereby limiting the efficiency of plant genetic engineering. Traditional DNA delivery methods for plants suffer from host-species limitations, low transformation efficiencies, tissue damage, or unavoidable and uncontrolled DNA integration into the host genome. We have demonstrated efficient plasmid DNA delivery into intact plants of several species with functionalized high-aspect-ratio carbon nanotube (CNT) nanoparticles (NPs), enabling efficient DNA delivery into a variety of non-model plant species (arugula, wheat, and cotton) and resulting in high protein expression levels without transgene integration. Herein, we provide a protocol that can be implemented by plant biologists and adapted to produce functionalized single-walled CNTs (SWNTs) with surface chemistries optimized for delivery of plasmid DNA in a plant species–independent manner. This protocol describes how to prepare, construct, and optimize polyethylenimine (PEI)-functionalized SWNTs and perform plasmid DNA loading. The authors also provide guidance on material characterization, gene expression evaluation, and storage conditions. The entire protocol, from the covalent functionalization of SWNTs to expression quantification, can be completed in 5 d.

Agriculture faces significant global challenges including climate change and an increasing food demand due to a growing population. Addressing these challenges will require the adoption of transformative innovations into biotechnology practice, such as nanotechnology. Recently, nanomaterials have emerged as unmatched tools for their use as biosensors, or as biomolecule delivery vehicles. Despite their increasingly prolific use, plant-nanomaterial interactions remain poorly characterized, drawing into question the breadth of their utility and their broader environmental compatibility. Herein, we characterize Arabidopsis thaliana transcriptional response to single walled carbon nanotubes (SWNTs) with two different surface chemistries commonly used for biosensing and nucleic acid delivery: oligonucleotide adsorbed-pristine SWNTs, and polyethyleneimine-SWNTs loaded with plasmid DNA (PEI-SWNTs), both introduced by leaf infiltration. We observed that SWNTs elicit a mild stress response almost undistinguishable from the infiltration process, indicating that these nanomaterials are well-tolerated by the plant. However, PEI-SWNTs induce a much larger transcriptional reprogramming that involves stress, immunity, and senescence responses. PEI-SWNT-induced transcriptional profile is very similar to that of mutant plants displaying a constitutive immune response or treated with stress-priming agrochemicals. We selected molecular markers from our transcriptomic analysis and identified PEI as the main cause of this reaction. We show that PEI-SWNT response is concentration-dependent and, when persistent over time, leads to cell death. We probed a panel of PEI variant-functionalized SWNTs across two plant species and identified biocompatible SWNT surface functionalizations. Our results highlight the importance of nanoparticle surface chemistry on their biocompatibility and will facilitate the use of functionalized nanomaterials for agricultural improvement.Significance statementNanomaterials can be used in agriculture as biosensors to monitor plant health, as fertilizers or growth regulators, and as delivery vehicles for genome engineering reagents to improve crops. However, the interactions between nanoparticles and plant cells are not well understood. Here, we characterize the plant transcriptomic response to single-walled carbon nanotubes (SWNTs) commonly used for sensing and nucleic acid delivery. While SWNTs themselves are well tolerated by plants, SWNTs surface-functionalized with positively charged polymers become toxic and produce cell death. We identify molecular markers of this toxic response to create biocompatible SWNT formulations. These results highlight the significance of nanoparticle surface chemistry, perhaps more than the nanoparticles themselves, on downstream interactions of nanoparticles with the environment.

Nanomaterials have emerged as an invaluable tool for the delivery of biomolecules such as DNA and RNA, with various applications in genetic engineering and post-transcriptional genetic manipulation. Alongside this development, there has been an increasing use of polymer-based techniques, such as polyethyleneimine (PEI), to electrostatically load polynucleotide cargoes onto nanomaterial carriers. However, there remains a need to assess nanomaterial properties, conjugation conditions, and biocompatibility of these nanomaterial-polymer constructs, particularly for use in plant systems. In this work, we develop mechanisms to optimize DNA loading on single-walled carbon nanotubes (SWNTs) with a library of polymer-SWNT constructs and assess DNA loading ability, polydispersity, and both chemical and colloidal stability. Counterintuitively, we demonstrate that polymer hydrolysis from nanomaterial surfaces can occur depending on polymer properties and attachment chemistries, and describe mitigation strategies against construct degradation. Given the growing interest in delivery applications in plant systems, we also assess the toxicity of polymer-based nanomaterials in plants and provide recommendations for future design of nanomaterial-based polynucleotide delivery strategies.

Plant synthetic biology requires precise characterization of genetic elements to construct complex genetic circuits that can improve plant traits or confer them new characteristics. Transcriptional reporter assays are essential to quantify the effect of gene expression regulator elements. Therefore, transcriptional reporter systems are a key tool in understanding control of gene expression in biology.In this work we construct and characterize a dual-color luciferase ratiometric reporter system that possesses several advantages over currently used reporters. It is ratiometric, reducing variability and increasing consistency between experiments; it is fast, as both reporters can be measured at the same time in a single reaction, and it is cheaper to perform than current dual-luciferase reporter assays. We have validated our system quantifying the transcriptional capability of a panel of promoters and terminators commonly used in synthetic biology with a broad range of expression magnitudes, and in a biologically relevant system, nitrate response.

Plants will benefit from genetic engineering to feed our growing global population in the midst of climate change, to provide our most clinically-relevant drugs, and for our environmental remediation efforts. New generation genome engineering technologies, such as CRISPR/Cas, hold the potential to alter plant genes in a precise and targeted manner. The first step of any plant genetic engineering application is to deliver important biomolecular cargoes into the desired subcellular locations of plant cells. However, the limitations of traditional plant gene delivery methods, i.e. Agrobacterium and gene gun, prevent plant genetic engineering from reaching its full potential. Given the lack of efficient biomolecule delivery vehicles to plants and potential impact of plant biotechnology on food security and sustainability, we developed nanoparticle-based platforms to efficiently deliver small interfering RNA1,2 and plasmid DNA3,4 cargoes into plant cells in a species-independent manner, which eliminates the use of gene gun and addresses the main delivery bottlenecks of plant genetic engineering. We show that pristine single walled carbon nanotubes (SWCNTs) can adsorb small interfering RNA molecules through pi-pi stacking for plant delivery, whereas a covalently modified and positively charged SWCNT platform is more efficient for large double-stranded DNA delivery to mature plant leaf cells. This talk will first briefly summarize the development, characterization and biological verification of each nanoparticle platform for plant delivery applications. Next, I will present our recent results of CRISPR/Cas gene editing in plant leaves using the SWCNT DNA delivery platform5. The talk will be concluded by discussing the several assays performed to demonstrate the effect of nanoparticles on plant health, including an RNA-sequencing analysis of nanoparticle-treated plant leaves6. We believe the SWCNT delivery technologies that are discussed in this talk will have an immense impact on food security and sustainability by enabling the generation of improved plants that can rapidly reach the market in a cost-effective manner. Demirer G.S.*, Zhang H.* et al., DNA Nanostructures Coordinate Gene Silencing in Mature Plants. PNAS (2019). Demirer G.S. et al., Carbon Nanocarriers Deliver siRNA to Intact Plant Cells for Efficient Gene Knockdown. Science Advances (2020). Demirer G.S. et al., High Aspect Ratio Nanomaterials Enable Delivery of Functional Genetic Material Without Transgenic DNA Integration in Mature Plants. Nature Nanotechnology (2019). Demirer G.S. et al., Carbon nanotube–mediated DNA delivery without transgene integration in intact plants. Nature Protocols (2019). Demirer G.S. et al., Nanotechnology to advance CRISPR–Cas genetic engineering of plants. Nature Nanotechnology (2021). González-Grandío E., Demirer, G.S. et al. Carbon nanotube biocompatibility in plants is determined by their surface chemistry. bioRxiv (2021).

The control of branch outgrowth is critical for plant fitness, stress resilience and crop yield. The Arabidopsis thaliana transcription factor BRANCHED1 (BRC1) plays a pivotal role in this process as it integrates signals that inhibit axillary bud growth to control shoot branching. Despite the remarkable activity of BRC1 as a potent growth inhibitor, the mechanisms by which it promotes and maintains bud dormancy are still largely unknown. Here we combine ChIP-seq, transcriptomic and systems biology approaches to characterise the BRC1-regulated gene network. We identify a group of BRC1 direct target genes encoding transcription factors (BTFs) that orchestrate, together with BRC1, an intricate transcriptional network enriched in abscisic acid signalling components. The BRC1 network is enriched in feed-forward loops and feed-back loops, robust against noise and mutation, reversible in response to stimuli, and stable once established. This knowledge is fundamental to adapt plant architecture and crop production to ever-changing environmental conditions. Dutch Scientific Organization NWO-JSTP 833.13.008 Ministerio de Economía y Competitividad BIO2014-57011-R Ministerio de Economía y Competitividad BIO2017-84363-R Ministerio de Economía y Competitividad BIO2017-84066-R

Targeted downregulation of select endogenous plant genes is known to confer disease or pest resistance in crops and is routinely accomplished via transgenic modification of plants for constitutive gene silencing. An attractive alternative to the use of transgenics or pesticides in agriculture is the use of a 'green' alternative known as RNAi, which involves the delivery of siRNAs that downregulate endogenous genes to confer resistance. However, siRNA is a molecule that is highly susceptible to enzymatic degradation and is difficult to deliver across the lignin-rich and multi-layered plant cell wall that poses the dominant physical barrier to biomolecule delivery in plants. We have demonstrated that DNA nanostructures can be utilized as a cargo carrier for direct siRNA delivery and gene silencing in mature plants. The size, shape, compactness and stiffness of the DNA nanostructure affect both internalization into plant cells and subsequent gene silencing efficiency. Herein, we provide a detailed protocol that can be readily adopted with standard biology benchtop equipment to generate geometrically optimized DNA nanostructures for transgene-free and force-independent siRNA delivery and gene silencing in mature plants. We further discuss how such DNA nanostructures can be rationally designed to efficiently enter plant cells and deliver cargoes to mature plants, and provide guidance for DNA nanostructure characterization, storage and use. The protocol described herein can be completed in 4 d.

Significance Shoot-branching patterns affect key aspects of plant life and are important targets for crop breeding. However, we are still ignorant of the genetic mechanisms controlling locally an important decision during branch development: whether the axillary bud grows out to give a lateral shoot or remains dormant. Here we show that the TEOSINTE BRANCHED1, CYCLOIDEA, PCF (TCP) transcriptional regulator BRANCHED1 (BRC1), which acts inside axillary buds, binds and activates three genes encoding Homeodomain leucine zipper (HD-ZIP) transcription factors. These factors, together with BRC1, trigger a cascade leading to local abscisic acid (ABA) accumulation and response, essential for bud dormancy under light-limiting conditions. This finding demonstrates a direct relationship between BRC1 and ABA signaling and places ABA downstream of BRC1 in the control of axillary bud dormancy.

In angiosperms, shoot branching greatly determines overall plant architecture and affects fundamental aspects of plant life. Branching patterns are determined by genetic pathways conserved widely across angiosperms. In Arabidopsis thaliana (Brassicaceae, Rosidae) BRANCHED1 (BRC1) plays a central role in this process, acting locally to arrest axillary bud growth. In tomato (Solanum lycopersicum, Solanaceae, Asteridae) we have identified two BRC1-like paralogues, SlBRC1a and SlBRC1b. These genes are expressed in arrested axillary buds and both are down-regulated upon bud activation, although SlBRC1a is transcribed at much lower levels than SlBRC1b. Alternative splicing of SlBRC1a renders two transcripts that encode two BRC1-like proteins with different C-t domains due to a 3'-terminal frameshift. The phenotype of loss-of-function lines suggests that SlBRC1b has retained the ancestral role of BRC1 in shoot branch suppression. We have isolated the BRC1a and BRC1b genes of other Solanum species and have studied their evolution rates across the lineages. These studies indicate that, after duplication of an ancestral BRC1-like gene, BRC1b genes continued to evolve under a strong purifying selection that was consistent with the conserved function of SlBRC1b in shoot branching control. In contrast, the coding sequences of Solanum BRC1a genes have evolved at a higher evolution rate. Branch-site tests indicate that this difference does not reflect relaxation but rather positive selective pressure for adaptation.

The Teosinte branched1/Cincinnata/proliferating cell factor (TCP) family is a group of genes encoding plant-specific transcription factors that share the so-called TCP domain, a 59-amino-acid stretch predicted to form a noncanonical basic helix–loop–helix (bHLH) structure. The TCP domain allows DNA binding and facilitates protein–protein interactions. TCP genes have been found across the plant kingdom, from green algae to eudicots. Gene duplication and diversification of the family members led to an increase in family size, so that more than 20 TCP genes are found in eudicots species. Two major clades (class I and II) of TCP proteins can be distinguished with slightly different TCP domains. In this chapter, we summarize recent work analyzing the evolution of the TCP family in angiosperms, as well as the biochemical role of TCP proteins as transcriptional regulators. We highlight the role of particular residues of the TCP domain in protein–DNA and protein–protein interactions, and discuss the involvement of other protein regions in their posttranslational regulation and function.

Plants interpret a decrease in the red to far-red light ratio (R:FR) as a sign of impending shading by neighboring vegetation. This triggers a set of developmental responses known as shade avoidance syndrome. One of these responses is reduced branching through suppression of axillary bud outgrowth. The Arabidopsis thaliana gene BRANCHED1 (BRC1), expressed in axillary buds, is required for branch suppression in response to shade. Unlike wild-type plants, brc1 mutants develop several branches after a shade treatment. BRC1 transcription is positively regulated 4 h after exposure to low R:FR. Consistently, BRC1 is negatively regulated by phytochrome B. Transcriptional profiling of wild-type and brc1 buds of plants treated with simulated shade has revealed groups of genes whose mRNA levels are dependent on BRC1, among them a set of upregulated abscisic acid response genes and two networks of cell cycle– and ribosome-related downregulated genes. The downregulated genes have promoters enriched in TEOSINTE BRANCHED1, CYCLOIDEA, and PCF (TCP) binding sites, suggesting that they could be transcriptionally regulated by TCP factors. Some of these genes respond to BRC1 in seedlings and buds, supporting their close relationship with BRC1 activity. This response may allow the rapid adaptation of plants to fluctuations in the ratio of R:FR light.

In angiosperms, shoot branching greatly determines overall plant architecture and affects fundamental aspects of plant life. Branching patterns are determined by genetic pathways conserved widely across angiosperms. In Arabidopsis thaliana (Brassicaceae, Rosidae) BRANCHED1 (BRC1) plays a central role in this process, acting locally to arrest axillary bud growth. In tomato (Solanum lycopersicum, Solanaceae, Asteridae) we have identified two BRC1-like paralogues, SlBRC1a and SlBRC1b. These genes are expressed in arrested axillary buds and both are down-regulated upon bud activation, although SlBRC1a is transcribed at much lower levels than SlBRC1b. Alternative splicing of SlBRC1a renders two transcripts that encode two BRC1-like proteins with different C-t domains due to a 3'-terminal frameshift. The phenotype of loss-of-function lines suggests that SlBRC1b has retained the ancestral role of BRC1 in shoot branch suppression. We have isolated the BRC1a and BRC1b genes of other Solanum species and have studied their evolution rates across the lineages. These studies indicate that, after duplication of an ancestral BRC1-like gene, BRC1b genes continued to evolve under a strong purifying selection that was consistent with the conserved function of SlBRC1b in shoot branching control. In contrast, the coding sequences of Solanum BRC1a genes have evolved at a higher evolution rate. Branch-site tests indicate that this difference does not reflect relaxation but rather positive selective pressure for adaptation.

In Arabidopsis, axillary buds may become dormant in response to a far-red rich light or to increased apical dominance. BRANCHED1 (BRC1) is required for this response. Transcriptional profiling studies of wild-type and brc1 mutant buds allowed the identification of sets of BRC1-dependent genes including a group of ABA-related genes upregulated in dormant buds. By using BRC1 inducible lines we demonstrate that 2 of these ABA response factors, ABF3 and ABI5, are positively regulated in axillary buds by BRC1 after induction. To get further insight into the genetic control of the growth-to-dormancy transition in buds we have also compared this transcriptomic data with 2 additional "active vs dormant bud" transcriptomic data sets and found "core" co-regulated gene networks tightly associated to each condition.