I. Khait, Ohad Lewin-Epstein, R. Sharon, K. Saban, R. Perelman, A. Boonman, Y. Yovel, L. Hadany

Stressed plants show altered phenotypes, including changes in color, smell, and shape. Yet, the possibility that plants emit airborne sounds when stressed – similarly to many animals – has not been investigated. Here we show, to our knowledge for the first time, that stressed plants emit airborne sounds that can be recorded remotely, both in acoustic chambers and in greenhouses. We recorded ∼65 dBSPL ultrasonic sounds 10 cm from tomato and tobacco plants, implying that these sounds could be detected by some organisms from up to several meters away. We developed machine learning models that were capable of distinguishing between plant sounds and general noises, and identifying the condition of the plants – dry, cut, or intact – based solely on the emitted sounds. Our results suggest that animals, humans, and possibly even other plants, could use sounds emitted by a plant to gain information about the plant’s condition. More investigation on plant bioacoustics in general and on sound emission in plants in particular may open new avenues for understanding plants and their interactions with the environment, and it may also have a significant impact on agriculture.

Amy Watson, Sreya Ghosh, Matthew J Williams, William S. Cuddy, James Simmonds, María-Dolores Rey, M. Asyraf Md. Hatta, Alison Hinchliffe, Andrew Steed, Daniel Reynolds, Nikolai Adamski, Andy Breakspear, Andrey Korolev, Tracey Rayner, Laura E. Dixon, Adnan Riaz, William Martin, Merrill Ryan, David Edwards, Jacqueline Batley, Raman Harsh, Christian Rogers, Claire Domoney, Graham Moore, Wendy Harwood, Paul Nicholson, Mark J. Dieters, Ian H. DeLacy, Ji Zhou, Cristobal Uauy, Scott Boden, Robert Fraser Park, Brande B. H. Wulff, Lee T. Hickey

The growing human population and a changing environment have raised significant concern for global food security, with the current improvement rate of several important crops inadequate to meet future demand [1]. This slow improvement rate is attributed partly to the long generation times of crop plants. Here we present a method called 'speed breeding', which greatly shortens generation time and accelerates breeding and research programs. Speed breeding can be used to achieve up to 6 generations per year for spring wheat (Triticum aestivum), durum wheat (T. durum), barley (Hordeum vulgare), chickpea (Cicer arietinum), and pea (Pisum sativum) and 4 generations for canola (Brassica napus), instead of 2-3 under normal glasshouse conditions. We demonstrate that speed breeding in fully-enclosed controlled-environment growth chambers can accelerate plant development for research purposes, including phenotyping of adult plant traits, mutant studies, and transformation. The use of supplemental lighting in a glasshouse environment allows rapid generation cycling through single seed descent and potential for adaptation to larger-scale crop improvement programs. Cost-saving through LED supplemental lighting is also outlined. We envisage great potential for integrating speed breeding with other modern crop breeding technologies, including high-throughput genotyping, genome editing, and genomic selection, accelerating the rate of crop improvement.

Gozde S. Demirer, Huan Zhang, Juliana L Matos, Natalie Goh, Francis Cunningham, Younghun Sung, Roger Chang, Abhishek J Aditham, Linda Chio, Myeong-Je Cho, Brian Staskawicz, Markita P Landry

Genetic engineering of plants is at the core of sustainability efforts, natural product synthesis, and agricultural crop engineering. The plant cell wall is a barrier that limits the ease and throughput with which exogenous biomolecules can be delivered to plants. Current delivery methods either suffer from host range limitations, low transformation efficiencies, tissue damage, or unavoidable DNA integration into the host genome. Here, we demonstrate efficient diffusion-based biomolecule delivery into tissues and organs of intact plants of several species with a suite of pristine and chemically-functionalized high aspect ratio nanomaterials. Efficient DNA delivery and strong protein expression without transgene integration is accomplished in Nicotiana benthamiana (Nb), Eruca sativa (arugula), Triticum aestivum (wheat) and Gossypium hirsutum (cotton) leaves and arugula protoplasts. We also demonstrate a second nanoparticle-based strategy in which small interfering RNA (siRNA) is delivered to Nb leaves and silence a gene with 95% efficiency. We find that nanomaterials not only facilitate biomolecule transport into plant cells but also protect polynucleotides from nuclease degradation. Our work provides a tool for species-independent and passive delivery of genetic material, without transgene integration, into plant cells for diverse biotechnology applications.

Tatiana Mitiouchkina, Alexander S Mishin, Louisa Gonzalez Somermeyer, Nadezhda M Markina, Tatiana V Chepurnyh, Elena B Guglya, Tatiana A Karataeva, Kseniia A Palkina, Ekaterina S Shakhova, Liliia I Fakhranurova, Sofia V Chekova, Aleksandra S Tsarkova, Yaroslav V Golubev, Vadim V Negrebetsky, Sergey A Dolgushin, Pavel V Shalaev, Olesya A Melnik, Victoria O Shipunova, Sergey M Deyev, Andrey I Bubyrev, Alexander S Pushin, Vladimir V Choob, Sergey V Dolgov, Fyodor Kondrashov, Ilia V. Yampolsky, Karen S. Sarkisyan

In contrast to fluorescent proteins, light emission from luciferase reporters requires exogenous addition of a luciferin substrate. Bacterial bioluminescence has been the single exception, where an operon of five genes is sufficient to produce light autonomously. Although commonly used in prokaryotic hosts, toxicity of the aldehyde substrate has limited its use in eukaryotes. Here we demonstrate autonomous luminescence in a multicellular eukaryotic organism by incorporating a recently discovered fungal bioluminescent system into tobacco plants. We monitored these light-emitting plants from germination to flowering, observing temporal and spatial patterns of luminescence across time scales from seconds to months. The dynamic patterns of luminescence reflected progression through developmental stages, circadian oscillations, transport, and response to injuries. As with other fluorescent and luminescent reporters, we anticipate that this system will be further engineered for varied purposes, especially where exogenous addition of substrate is undesirable.

Cara Doyle, Katie Higginbottom, Thomas A. Swift, Mark Winfield, Christopher Bellas, David Benito-Alifonso, Taryn Fletcher, M. Carmen Galan, Keith Edwards, Heather M Whitney

Potential innovation in Plant research using gene-edited and genetically modified plants is currently being hindered by inefficient and costly plant transformation. We show that carbon dots formed from natural materials (quasi-spherical, <10nm nanoparticles) can act as a fast vehicle for carrying plasmids into mature plant cells, resulting in transient plant transformation in a number of important crop species with no negative impacts on photosynthesis or growth. We further show that GFP, Cas9, and gRNA introduced into wheat via foliar application (spraying on) of plasmid coated carbon dots are expressed and, in the case of Cas9, make genome edits in SPO11 genes. Therefore, we present a protocol for spray-on gene editing that is simple, inexpensive, fast, transforms in planta , and is applicable to multiple crop species. We believe this technique creates many opportunities for the future of plant transformation in research and shows great promise for plant protein production systems.

Anton Sharakshane

Dependencies have been shown and conversion factors have been determined, which allow to estimate PPFD, YPFD and radiometric power density of white LED light according to the known illumination in lux. A technique for estimating photosynthetically active radiation, which has an adequate accuracy for the task of illuminating plants, has been determined.

Daniela Paula de Toledo Thomazella, Quinton Brail, Douglas Dahlbeck, Brian Staskawicz

Pathogenic microbes are responsible for severe production losses in crops worldwide. The use of disease resistant crop varieties can be a sustainable approach to meet the food demand of the world's growing population. However, classical plant breeding is usually laborious and time-consuming, thus hampering efficient improvement of many crops. With the advent of genome editing technologies, in particular the CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-Cas9) system, we are now able to introduce improved crop traits in a rapid and efficient manner. In this work, we genome edited durable disease resistance in tomato by modifying a specific gene associated with disease resistance. Recently, it was demonstrated that inactivation of a single gene called DMR6 (downy mildew resistance 6) confers resistance to several pathogens in Arabidopsis thaliana. This gene is specifically up-regulated during pathogen infection, and mutations in the dmr6 gene results in increased salicylic acid levels. The tomato SlDMR6-1 orthologue Solyc03g080190.2 is also up-regulated during infection by Pseudomonas syringae pv. tomato and Phytophthora capsici. Using the CRISPR/Cas-9 system, we generated tomato plants with small deletions in the SlDMR6-1 gene that result in frameshift and premature truncation of the protein. Remarkably, these mutants do not have significant detrimental effects in terms of growth and development under greenhouse conditions and show disease resistance against different pathogens, including P. syringae, P. capsici and Xanthomonas spp.

Tae-Wuk Kim, Chan Ho Park, Chuan-Chih Hsu, Jia-Ying Zhu, Yuchun Hsiao, Tess Branon, Shouling Xu, Alice Y Ting, Zhi-Yong Wang

Transient protein-protein interactions (PPIs), such as those between posttranslational modifying enzymes and their substrates, play key roles in cellular regulation, but are difficult to identify. Here we demonstrate the application of enzyme-catalyzed proximity labeling (PL), using the engineered promiscuous biotin ligase TurboID, as a sensitive method for characterizing PPIs in signaling networks. We show that TurboID fused with the GSK3-like kinase BIN2 or a PP2A phosphatase biotinylates their known substrate, the BZR1 transcription factor, with high specificity and efficiency. We optimized the protocol of biotin labeling and affinity purification in transgenic Arabidopsis expressing a BIN2-TurboID fusion protein. Subsequent quantitative mass spectrometry (MS) analysis identified about three hundred proteins biotinylated by BIN2-TurboID more efficiently than the YFP-TurboID control. These include a significant subset of previously proven BIN2 interactors and a large number of new BIN2-proximal proteins that uncover a broad BIN2 signaling network. Our study illustrates that PL-MS using TurboID is a powerful tool for mapping signaling networks, and reveals broad roles of BIN2 kinase in cellular signaling and regulation in plants.

Ryan C Lynch, Daniela Vergara, Silas Tittes, Kristin White, C.J. Schwartz, Matthew J Gibbs, Travis C Ruthenburg, Kymron deCesare, Donald P Land, Nolan C Kane

Plants of the Cannabis genus are the only producers of phytocannabinoids, terpenoid compounds that strongly interact with evolutionarily ancient endocannabinoid receptors shared by most bilaterian taxa. For millennia, the plant has been cultivated for these compounds, but also for food, rope, paper, and clothing. Today, specialized varieties yielding high-quality textile fibers, nutritional seed oil or high cannabinoid content are cultivated across the globe. However, the genetic identities and histories of these diverse populations remain largely obscured. We analyzed the nuclear genomic diversity among 340 Cannabis varieties, including fiber and seed oil hemp, high cannabinoid drug-types and feral populations. These analyses demonstrate the existence of at least three major groups of diversity, with European hemp varieties more closely related to narrow leaflet drug-types (NLDT) than to broad leaflet drug-types (BLDT). The BLDT group appears to encompass less diversity than the NLDT, which reflects the larger geographic range of NLDTs, and suggests a more recent origin of domestication of the BLDTs. As well as being genetically distinct, hemp, NLDT and BLDT genetic groups each produce unique cannabinoid and terpenoid content profiles. This combined analysis of population genomic and trait variation informs our understanding of the potential uses of different genetic variants for medicine and agriculture, providing valuable insights and tools for a rapidly emerging, valuable legal industry

Sreya Ghosh, Amy Watson, Oscar E. Gonzalez-Navarro, Ricardo Ramírez-González, Luis Yanes, Marcela Mendoza-Suárez, James Simmonds, Rachel Wells, Tracey Rayner, Phon Green, Amber Hafeez, Sadiye Hayta, Rachel E. Melton, Andrew Steed, Abhimanyu Sarkar, Jeremy Carter, Lionel Perkins, John Lord, Mark Tester, Anne Osbourn, Matthew James Moscou, Paul Nicholson, Wendy Harwood, Cathie Martin, Claire Domoney, Cristobal Uauy, Brittany Hazard, Brande B. H. Wulff, Lee T. Hickey

To meet the challenge of feeding a growing population, breeders and scientists are continuously looking for ways to increase genetic gain in crop breeding. One way this can be achieved is through 'speed breeding' (SB), which shortens the breeding cycle and accelerates research studies through rapid generation advancement. The SB method can be carried out in a number of ways, one of which involves extending the duration of a plant's daily exposure to light (photoperiod) combined with early seed harvest in order to cycle quickly from seed to seed, thereby reducing the generation times for some long-day (LD) or day-neutral crops. Here we present glasshouse and growth chamber-based SB protocols with supporting data from experimentation with several crop species. These protocols describe the growing conditions, including soil media composition, lighting, temperature and spacing, which promote rapid growth of spring and winter bread wheat, durum wheat, barley, oat, various members of the Brassica family, chickpea, pea, grasspea, quinoa and the model grass Brachypodium distachyon. Points of flexibility within the protocols are highlighted, including how plant density can be increased to efficiently scale-up plant numbers for single seed descent (SSD) purposes. Conversely, instructions on how to perform SB on a small-scale by creating a benchtop SB growth cabinet that enables optimization of parameters at a low cost are provided. We also outline the procedure for harvesting and germinating premature wheat, barley and pea seed to reduce generation time. Finally, we provide troubleshooting suggestions to avoid potential pitfalls.

Akihiro Shimokawa, Yuki Tonooka, Misato Matsumoto, Hironori Ara, Hiroshi Suzuki, Naoki Yamauchi, Masayoshi Shigyo

Because global climate change has made agricultural supply unstable, plant factories are expected to be a safe and stable means of food production. As the light source of a plant factory or controlled greenhouse, the light emitting diode (LED) is expected to solve cost problems and promote plant growth efficiently. In this study, we examined the light condition created by using monochromatic red and blue LEDs, to provide both simultaneous and alternating irradiation to leaf lettuce. The result was that simultaneous red and blue irradiation promoted plant growth more effectively than monochromatic and fluorescent light irradiation. Moreover, alternating red and blue light accelerated plant growth significantly even when the total light intensity per day was the same as with simultaneous irradiation. The fresh weight in altering irradiation was almost two times higher than with fluorescent light and about 1.6 times higher than with simultaneous irradiation. The growth-promoting effect of alternating irradiation of red and blue light was observed in different cultivars. From the results of experiments, we offer a novel plant growth method named "Shigyo Method", the core concept of which is the alternating irradiation of red and blue light.

Serena R.G. Page, Adrian S Monthony, A. Maxwell P. Jones

Micropropagation of Cannabis sativa is an emerging area for germplasm storage and large-scale production of clean plants. Existing protocols use a limited number of genotypes and are often not reproducible. Previous studies reported MS + 0.5 μM TDZ to be optimal for Cannabis nodal micropropagation, yet our preliminary studies using nodal explants suggested this media may not be optimal. It resulted in excessive callus formation, hyperhydricity, low multiplication rates, and high mortality rates. Following an initial screen of four commonly used basal salt mixtures (MS, B5, BABI, and DKW), we determined that DKW produced the healthiest plants. In a second experiment, the multiplication rate and canopy area of explants grown on MS + 0.5 μM TDZ and DKW + 0.5 μM TDZ were compared using five drug-type cultivars to determine if the preference for DKW was genotype-dependent. Four cultivars had significantly higher multiplication rates on DKW + 0.5 μM TDZ with the combined average being 1.5x higher than explants grown on MS + 0.5 μM TDZ. The canopy area was also significantly larger on DKW + 0.5 μM TDZ for four cultivars with the combined average being twice as large as the explants grown on MS + 0.5 μM TDZ. In the third experiment, callogenesis was compared using a range of 2,4-D concentrations (0-30 μM) on both MS and DKW and similarly, callus growth was superior on DKW. This study presents the largest comparison of basal salt compositions on the micropropagation of five commercially grown Cannabis cultivars to date. ### Competing Interest Statement The authors have declared no competing interest. * 2,4-D : 2,4 dichlorophenoxyacetic acid ANOVA : analysis of variance BABI : BDS as modified at Arkansas Bioscience Institute CBD : cannabidiol CCDB : Canadian Centre for DNA Barcoding DKW : Driver and Kuniyuki Walnut MS : Murashige and Skoog NIDA : National Institute on Drug Abuse PAR : photosynthetically active radiation PGR : plant growth regulator PPM : Plant Preservative Mixture SSR : simple sequence repeats TDZ : thidiazuron THC : Δ9-tetrahydrocannabinol

Margaret H Frank, Daniel H Chitwood

Chimeras, organisms that are composed of cells of more than one genotype, captured the human imagination long before they were formally described and used in the laboratory. These organisms owe their namesake to a fire-breathing monster from Greek mythology that has the head of a lion, the body of a goat, and the tail of a serpent. The first description of a non-fictional chimera dates back to the middle of the seventeenth century when the Florentine gardener Pietro Nati discovered an adventitious shoot growing from the graft junction between sour orange (Citrus aurantium) and citron (C. medica). This perplexing chimera that grows with sectors phenotypically resembling each of the citrus progenitors inspired discussion and wonder from the scientific community and was fittingly named the Bizzaria. Initially, the Bizzaria was believed to be an asexual hybrid that formed from a cellular fusion between the grafted parents; however, in-depth cellular analyses carried out centuries later demonstrated that the Bizzaria, along with other chimeras, owe their unique sectored appearance to a conglomeration of cells from the two donors. Since this pivotal discovery at the turn of the twentieth century, chimeras have served both as tools and as unique biological phenomena that have contributed to our understanding of plant development at the cellular, tissue, and organismal level. Rapid advancements in genome sequencing technologies have enabled the establishment of new model species with novel morphological and developmental features that enable the generation of chimeric organisms. In this review, we show that genetic mosaic and chimera studies provide a technologically simple way to delve into the organismal, genetic, and genomic inner workings underlying the development of diverse model organisms. Moreover, we discuss the unique opportunity that chimeras present to explore universal principles governing intercellular communication and the coordination of organismal biology in a heterogenomic landscape.

Anton Sharakshane

The highest intensity of photosynthesis is obtained under red light, but plants die or their growth gets disrupted if only red light is used. For example, Korean researchers have shown that under pure red light the amount of the grown lettuce is greater than under a combination of red and blue light, but the leaves have a significantly smaller amount of chlorophyll, polyphenols and antioxidants. And the researchers at the Faculty of Biology of the Moscow State University have found that the synthesis of sugars is reduced, growth is inhibited and no blossoming occurs in the leaves of Chinese cabbage under narrow-band red and blue light (as compared to a sodium lamp). What kind of lighting is needed to get a fully developed, large, fragrant and tasty plant with moderate energy consumption?

Douglas Smith

Cannabinoid expression is an important genetically determined feature of cannabis that presents clinical and legal implications for patients seeking cannabinoid specific therapies like Cannabidiol (CBD). Cannabinoid, terpenoid, and flavonoid marker assisted selection can accelerate breeding efforts by offering genetic tools to select for desired traits at an early stage in growth. To this end, multiple models for chemotype inheritance have been described suggesting a complex picture for chemical phenotype determination. Here we explore the potential role of copy number variation of THCA Synthase using phased single molecule sequencing and demonstrate that copy number and sequence variation of this gene is common and suggests a more nuanced view of chemotype prediction.

Yuan-Yeu Yau, Mona Easterling, Lindsey Brennan

Tobacco is a model plant for genetic transformation, with leaf-disk transformation being the most commonly used method for its transformation. One disadvantage of leaf-disk transformation is obtaining an adequately sized leaf. Cotyledons from young seedlings are considered too small and fragile to use. In an attempt to overcome this drawback, a protocol was developed using toothpicks as a tool to inoculate cotyledons ~2mm in diameter. Agrobacterium tumefaciens LBA4404 hosting two different plasmids (pC35.BNK.2 or pRB140-Bxb1-op) was used for transformation. Fifty-six putative transgenic shoots (T0) were obtained from pC35.BNK.2 transformation. Among them, 38 (68%) grew roots in kanamycin-containing medium. Approximately, 35% of transgenic lines contained a single-copy transgenic locus based on Mendelian inheritance analysis and chi-square (χ2) test of T1 seedlings from 17 lines. To simplify the protocol, water-prepared Agrobacterium inoculum was used in pRB140-Bxb1-op (containing gus gene) transformation. This resulted in ~35% putative T0 transgenic lines stained strong blue with GUS histochemical staining assay. Both sets of results demonstrate toothpick inoculation to be an effective approach for Agrobacterium-mediated tobacco cotyledon transformation. This reduces wait time required in existing leaf-disk transformation method using mature leaves. Removal of step requiring submersion of explants in Agrobacterium liquid culture, the protocol also has advantages by minimizing Agrobacterium overgrowth and maintaining explant fitness for later tissue-culturing.

Christine N Shulse, Benjamin Cole, Gina M Turco, Yiwen Zhu, Siobhan M Brady, Diane E. Dickel

Single-cell transcriptome analysis of heterogeneous tissues can provide high-resolution windows into the genomic basis and spatiotemporal dynamics of developmental processes. Here we demonstrate the feasibility of high-throughput single-cell RNA sequencing of plant tissue using the Drop-seq approach. Profiling of >4,000 individual cells from the Arabidopsis root provides transcriptomes and marker genes for a diversity of cell types and illuminates the gene expression changes that occur across endodermis development.

Bruno Pok Man Ngou, Hee-Kyung Ahn, Pingtao Ding, Jonathan DG Jones

The plant immune system involves cell-surface receptors that detect intercellular pathogen-derived molecules, and intracellular receptors that activate immunity upon detection of pathogen-secreted effectors that act inside the plant cell. Surface receptor-mediated immunity has been extensively studied but in authentic interactions between plants and microbial pathogens, its presence impedes study of intracellular receptor-mediated immunity alone. How these two immune pathways interact is poorly understood. Here, we reveal mutual potentiation between these two recognition-dependent defense pathways. Recognition by surface receptors activates multiple protein kinases and NADPH oxidases, whereas intracellular receptors primarily elevate abundance of these proteins. Reciprocally, the intracellular receptor-dependent hypersensitive cell death response is strongly enhanced by activation of surface receptors. Activation of either immune system alone is insufficient to provide effective resistance against Pseudomonas syringae. Thus, immune pathways activated by cell-surface and intracellular receptors mutually potentiate to activate strong defense that thwarts pathogens. By studying the activation of intracellular receptors in the absence of surface receptor-mediated immunity, we have dissected the relationship between the two distinct immune systems. These findings reshape our understanding of plant immunity and have broad implications for crop improvement. ### Competing Interest Statement The authors have declared no competing interest.

Jiorgos Kourelis, Clemence Marchal, Sophien Kamoun

Plant pathogens cause recurrent epidemics that threaten crop yield and global food security. Efforts to retool the plant immune system have been limited to modifying natural components and can be nullified by the emergence of new pathogen races. Therefore, there is a need to develop made-to-order synthetic plant immune receptors with resistance tailored to the pathogen genotypes present in the field. Here we show that plant immune receptors can be used as scaffolds for VHH nanobody fusions that bind fluorescent proteins (FPs). The receptor-nanobody fusions signal in the presence of the corresponding FP and confer resistance against plant viruses expressing FPs. Given that nanobodies can be raised against virtually any molecule, immune receptor-nanobody fusions have the potential to generate resistance against all major plant pathogens and pests.

Brieanne Vaillancourt, C. Robin Buell

The ability to generate long reads on the Oxford Nanopore Technologies sequencing platform is dependent on the isolation of high molecular weight DNA free of impurities. For some taxa, this is relatively straightforward; however, for plants, the presence of cell walls and a diverse set of specialized metabolites such as lignin, phenolics, alkaloids, terpenes, and flavonoids present significant challenges in the generation of DNA suitable for production of long reads. Success in generating long read lengths and genome assemblies of plants has been reported using diverse DNA isolation methods, some of which were tailored to the target species and/or required extensive labor. To avoid the need to optimize DNA isolation for each species, we developed a taxa-independent DNA isolation method that is relatively simple and efficient. This method expands on the Oxford Nanopore Technologies high molecular weight genomic DNA protocol from plant leaves and utilizes a conventional cetyl trimethylammonium bromide extraction followed by removal of impurities and short DNA fragments using commercially available kits that yielded robust N50 read lengths and yield on Oxford Nanopore Technologies flow cells. * CTAB : cetyl trimethylammonium bromide ONT : Oxford Nanopore Technologies