Carbon Nanomaterial Fluorescent Probes and Their Biological Applications.

Fluorescent carbon nanomaterials have broadly useful chemical and photophysical attributes that are conducive to applications in biology. In this review, we focus on materials whose photophysics allow for the use of these materials in biomedical and environmental applications, with emphasis on imaging, biosensing, and cargo delivery. The review focuses primarily on graphitic carbon nanomaterials including graphene and its derivatives, carbon nanotubes, as well as carbon dots and carbon nanohoops. Recent advances in and future prospects of these fields are discussed at depth, and where appropriate, references to reviews pertaining to older literature are provided.

Engineering agricultural soil microbiomes and predicting plant phenotypes.

Plant growth-promoting rhizobacteria (PGPR) can improve crop yields, nutrient use efficiency, plant tolerance to stressors, and confer benefits to future generations of crops grown in the same soil. Unlocking the potential of microbial communities in the rhizosphere and endosphere is therefore of great interest for sustainable agriculture advancements. Before plant microbiomes can be engineered to confer desirable phenotypic effects on their plant hosts, a deeper understanding of the interacting factors influencing rhizosphere community structure and function is needed. Dealing with this complexity is becoming more feasible using computational approaches. In this review, we discuss recent advances at the intersection of experimental and computational strategies for the investigation of plant-microbiome interactions and the engineering of desirable soil microbiomes.

Phosphate deprivation-induced changes in tomato are mediated by an interaction between brassinosteroid signaling and zinc.

Inorganic phosphate (Pi) is a necessary macronutrient for basic biological processes. Plants modulate their root system architecture (RSA) and cellular processes to adapt to Pi deprivation albeit with a growth penalty. Excess application of Pi fertilizer, on the contrary, leads to eutrophication and has a negative environmental impact. We compared RSA, root hair elongation, acid phosphatase activity, metal ion accumulation, and brassinosteroid hormone levels of Solanum lycopersicum (tomato) and Solanum pennellii, which is a wild relative of tomato, under Pi sufficiency and deficiency conditions to understand the molecular mechanism of Pi deprivation response in tomato. We showed that S. pennellii is partially insensitive to phosphate deprivation. Furthermore, it mounts a constitutive response under phosphate sufficiency. We demonstrate that activated brassinosteroid signaling through a tomato BZR1 ortholog gives rise to the same constitutive phosphate deficiency response, which is dependent on zinc overaccumulation. Collectively, these results reveal an additional strategy by which plants can adapt to phosphate starvation.

Synthetic biology for plant genetic engineering and molecular farming.

Many efforts have been put into engineering plants to improve crop yields and stress tolerance and boost the bioproduction of valuable molecules. Yet, our capabilities are still limited due to the lack of well-characterized genetic building blocks and resources for precise manipulation and given the inherently challenging properties of plant tissues. Advancements in plant synthetic biology can overcome these bottlenecks and release the full potential of engineered plants. In this review, we first discuss the recently developed plant synthetic elements from single parts to advanced circuits, software, and hardware tools expediting the engineering cycle. Next, we survey the advancements in plant biotechnology enabled by these recent resources. We conclude the review with outstanding challenges and future directions of plant synthetic biology.

Improving crop genetic transformation to feed the world.

Food security is threatened by rising global population and effects of climate change. Most of our calories come from a few crops that are difficult to improve. Lowe et al. developed a plant transformation approach enabling crop genetic engineering that could provide a route to a future with greater food security.

Plant biomacromolecule delivery methods in the 21st century.

The 21st century witnessed a boom in plant genomics and gene characterization studies through RNA interference and site-directed mutagenesis. Specifically, the last 15 years marked a rapid increase in discovering and implementing different genome editing techniques. Methods to deliver gene editing reagents have also attempted to keep pace with the discovery and implementation of gene editing tools in plants. As a result, various transient/stable, quick/lengthy, expensive (requiring specialized equipment)/inexpensive, and versatile/specific (species, developmental stage, or tissue) methods were developed. A brief account of these methods with emphasis on recent developments is provided in this review article. Additionally, the strengths and limitations of each method are listed to allow the reader to select the most appropriate method for their specific studies. Finally, a perspective for future developments and needs in this research area is presented.

GLRs: Mediating a defense-regeneration tradeoff in plants.

In this issue of Developmental Cell, Hernández-Coronado et al. present genetic and pharmacological evidence that reveals the central role of plant glutamate receptor-like proteins (GLRs) in the tradeoff between wounding-triggered regeneration and defense, offering new strategies to improve plant regeneration.

Toolboxes for plant systems biology research.

The terms 'systems' and 'synthetic biology' are often used together, with most scientists striding between the two fields rather than adhering to a single side. Often too, scientists want to understand a system to inform the design of gene circuits that could endow it with new functions. However, this does not need to be the progression of research, as synthetic constructs can help improve our understanding of a system. Here, we review synthetic biology tool kits with the potential to overcome pleiotropic effects, compensatory mechanisms, and redundancy in plants. Combined with -omics techniques, these tools could reveal novel insights on plant growth and development, an aim that has gained renewed urgency given the impact of climate change on crop productivity.

Nanoparticle cellular internalization is not required for RNA delivery to mature plant leaves.

Rapidly growing interest in the nanoparticle-mediated delivery of DNA and RNA to plants requires a better understanding of how nanoparticles and their cargoes translocate in plant tissues and into plant cells. However, little is known about how the size and shape of nanoparticles influence transport in plants and the delivery efficiency of their cargoes, limiting the development of nanotechnology in plant systems. In this study we employed non-biolistically delivered DNA-modified gold nanoparticles (AuNPs) of various sizes (5-20 nm) and shapes (spheres and rods) to systematically investigate their transport following infiltration into Nicotiana benthamiana leaves. Generally, smaller AuNPs demonstrated more rapid, higher and longer-lasting levels of association with plant cell walls compared with larger AuNPs. We observed internalization of rod-shaped but not spherical AuNPs into plant cells, yet, surprisingly, 10 nm spherical AuNPs functionalized with small-interfering RNA (siRNA) were the most efficient at siRNA delivery and inducing gene silencing in mature plant leaves. These results indicate the importance of nanoparticle size in efficient biomolecule delivery and, counterintuitively, demonstrate that efficient cargo delivery is possible and potentially optimal in the absence of nanoparticle cellular internalization. Overall, our results highlight nanoparticle features of importance for transport within plant tissues, providing a mechanistic overview of how nanoparticles can be designed to achieve efficacious biocargo delivery for future developments in plant nanobiotechnology.

Carbon nanotube biocompatibility in plants is determined by their surface chemistry.

BACKGROUND: 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. RESULTS: Herein, we characterize the response of Arabidopsis thaliana to single walled carbon nanotube (SWNT) exposure 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 pristine 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 adverse 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. CONCLUSIONS: While SWNTs themselves are well tolerated by plants, SWNTs surface-functionalized with positively charged polymers become toxic and produce cell death. We use molecular markers to identify more biocompatible SWNT formulations. Our results highlight the importance of nanoparticle surface chemistry on their biocompatibility and will facilitate the use of functionalized nanomaterials for agricultural improvement.

A Ratiometric Dual Color Luciferase Reporter for Fast Characterization of Transcriptional Regulatory Elements in Plants.

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.

Gold-Nanocluster-Mediated Delivery of siRNA to Intact Plant Cells for Efficient Gene Knockdown.

RNA interference, which involves the delivery of small interfering RNA (siRNA), has been used to validate target genes, to understand and control cellular metabolic pathways, and to use as a "green" alternative to confer pest tolerance in crops. Conventional siRNA delivery methods such as viruses and Agrobacterium-mediated delivery exhibit plant species range limitations and uncontrolled DNA integration into the plant genome. Here, we synthesize polyethylenimine-functionalized gold nanoclusters (PEI-AuNCs) to mediate siRNA delivery into intact plants and show that these nanoclusters enable efficient gene knockdown. We further demonstrate that PEI-AuNCs protect siRNA from RNase degradation while the complex is small enough to bypass the plant cell wall. Consequently, AuNCs enable gene knockdown with efficiencies of up 76.5 ± 5.9% and 76.1 ± 9.5% for GFP and ROQ1, respectively, with no observable toxicity. Our data suggest that AuNCs can deliver siRNA into intact plant cells for broad applications in plant biotechnology.

Efficient Transient Gene Knock-down in Tobacco Plants Using Carbon Nanocarriers.

Gene knock-down in plants is a useful approach to study genotype-phenotype relationships, render disease resistance to crops, and enable efficient biosynthesis of molecules in plants. Small interfering RNA (siRNA)-mediated gene silencing is one of the most common ways to achieve gene knock-down in plants. Traditionally, siRNA is delivered into intact plant cells by coding the siRNA sequences into DNA vectors, which are then delivered through viral and/or bacterial methods. In this protocol, we provide an alternative direct delivery method of siRNA molecules into intact plant cells for efficient transient gene knock-down in model tobacco plant, Nicotiana benthamiana, leaves. Our approach uses one dimensional carbon-based nanomaterials, single-walled carbon nanotubes (SWNTs), to deliver siRNA, and does not rely on viral/bacterial delivery. The distinct advantages of our method are i) there is no need for DNA coding of siRNA sequences, ii) this abiotic method could work in a broader range of plant species than biotic methods, and iii) there are fewer regulatory complications when using abiotic delivery methods, whereby gene silencing is transient without permanent modification of the plant genome. Graphic abstract.

Nanotechnology to advance CRISPR-Cas genetic engineering of plants.

CRISPR-Cas genetic engineering of plants holds tremendous potential for providing food security, battling biotic and abiotic crop stresses caused by climate change, and for environmental remediation and sustainability. Since the discovery of CRISPR-Cas technology, its usefulness has been demonstrated widely, including for genome editing in plants. Despite the revolutionary nature of genome-editing tools and the notable progress that these tools have enabled in plant genetic engineering, there remain many challenges for CRISPR applications in plant biotechnology. Nanomaterials could address some of the most critical challenges of CRISPR genome editing in plants through improvements in cargo delivery, species independence, germline transformation and gene editing efficiency. This Perspective identifies major barriers preventing CRISPR-mediated plant genetic engineering from reaching its full potential, and discusses ways that nanoparticle technologies can lower or eliminate these barriers. We also describe advances that are needed in nanotechnology to facilitate and accelerate plant genome editing. Timely advancement of the application of CRISPR technologies in plant engineering is crucial for our ability to feed and sustain the growing human population under a changing global climate.

Engineering DNA nanostructures for siRNA delivery in plants.

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.

Carbon nanocarriers deliver siRNA to intact plant cells for efficient gene knockdown.

Posttranscriptional gene silencing (PTGS) is a powerful tool to understand and control plant metabolic pathways, which is central to plant biotechnology. PTGS is commonly accomplished through delivery of small interfering RNA (siRNA) into cells. Standard plant siRNA delivery methods (Agrobacterium and viruses) involve coding siRNA into DNA vectors and are only tractable for certain plant species. Here, we develop a nanotube-based platform for direct delivery of siRNA and show high silencing efficiency in intact plant cells. We demonstrate that nanotubes successfully deliver siRNA and silence endogenous genes, owing to effective intracellular delivery and nanotube-induced protection of siRNA from nuclease degradation. This study establishes that nanotubes could enable a myriad of plant biotechnology applications that rely on RNA delivery to intact cells.

Nanobiolistics: An Emerging Genetic Transformation Approach.

Biolistic delivery of biomolecular cargoes to plants with micron-scale projectiles is a well-established technique in plant biotechnology. However, the relatively large micron-scale biolistic projectiles can result in tissue damage, low regeneration efficiency, and create difficulties for the biolistic transformation of isomorphic small cells or subcellular target organelles (i.e., mitochondria and plastids). As an alternative to micron-sized carriers, nanomaterials provide a promising approach for biomolecule delivery to plants. While most studies exploring nanoscale biolistic carriers have been carried out in animal cells and tissues, which lack a cell wall, we can nonetheless extrapolate their utility for nanobiolistic delivery of biomolecules in plant targets. Specifically, nanobiolistics has shown promising results for use in animal systems, in which nanoscale projectiles yield lower levels of cell and tissue damage while maintaining similar transformation efficiencies as their micron-scale counterparts. In this chapter, we specifically discuss biolistic delivery of nanoparticles for plant genetic transformation purposes and identify the figures of merit requiring optimization for broad-scale implementation of nanobiolistics in plant genetic transformations.

Carbon nanotube-mediated DNA delivery without transgene integration in intact plants.

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.