Search

Anti-CRISPR proteins are very efficient for inhibiting CRISPR/Cas9-based genome editing tools in both herbaceous and woody plant species.

Deciduous woody plants like poplar (Populus spp.) have seasonal bud dormancy. It has been challenging to simultaneously delay the onset of bud dormancy in the fall and advance bud break in the spring, as bud dormancy, and bud break were thought to be controlled by different genetic factors. Here, we demonstrate that heterologous expression of the REVEILLE1 gene (named AaRVE1) from Agave (Agave americana) not only delays the onset of bud dormancy but also accelerates bud break in poplar in field trials. AaRVE1 heterologous expression increases poplar biomass yield by 166% in the greenhouse. Furthermore, we reveal that heterologous expression of AaRVE1 increases cytokinin contents, represses multiple dormancy-related genes, and up-regulates bud break-related genes, and that AaRVE1 functions as a transcriptional repressor and regulates the activity of the DORMANCY-ASSOCIATED PROTEIN 1 (DRM1) promoter. Our findings demonstrate that AaRVE1 appears to function as a regulator of bud dormancy and bud break, which has important implications for extending the growing season of deciduous trees in frost-free temperate and subtropical regions to increase crop yield.

The genus Populus has long been used for environmental, agroforestry and industrial applications worldwide. Today Populus is also recognized as a desirable crop for biofuel production and a model tree for physiological and ecological research. As such, various modern biotechnologies, including CRISPR/Cas9-based techniques, have been actively applied to Populus for genetic and genomic improvements for traits such as increased growth rate and tailored lignin composition. However, CRISPR/Cas9 has been primarily used as the active Cas9 form to create knockouts in the hybrid poplar clone ‘717-1B4’ (Populus tremula x P. alba clone INRA 717-1B4). Alternative CRISPR/Cas9-based technologies, e.g., those involving modified Cas9 for gene activation and base editing, have not been evaluated in most Populus species for their efficacy. Here we employed a deactivated Cas9 (dCas9)-based CRISPR activation (CRISPRa) technique to fine-tune the expression of two target genes, TPX2 and LecRLK-G which play important roles in plant growth and defense response, in hybrid poplar clone ‘717-1B4’ and poplar clone ‘WV94’ (P. deltoides ‘WV94’), respectively. We observed that CRISPRa resulted in 1.2-fold to 7.0-fold increase in target gene expression through transient expression in protoplasts and Agrobacterium-mediated stable transformation, demonstrating the effectiveness of dCas9-based CRISPRa system in Populus. In addition, we applied Cas9 nickase (nCas9)-based cytosine base editor (CBE) to precisely introduce premature stop codons via C-to-T conversion, with an efficiency of 13%-14%, in the target gene PLATZ which encodes a transcription factor involved in plant fungal pathogen response in poplar clone ‘717-1B4’. Overall, we showcase the successful application of CRISPR/Cas-based technologies in gene expression regulation and precise gene engineering in two Populus species, facilitating the adoption of emerging genome editing tools in woody species.

The chromatin-based rule governing the selection and activation of replication origins remains to be elucidated. It is believed that DNA replication initiates from open chromatin domains; thus, replication origins reside in open and active chromatin. However, we report here that lysine-specific demethylase 1 (LSD1), which biochemically catalyzes H3K4me1/2 demethylation favoring chromatin condensation, interacts with the DNA replication machinery in human cells. We find that LSD1 level peaks in early S phase, when it is required for DNA replication by facilitating origin firing in euchromatic regions. Indeed, euchromatic zones enriched in H3K4me2 are the preferred sites for the pre-replicative complex (pre-RC) binding. Remarkably, LSD1 deficiency leads to a genome-wide switch of replication from early to late. We show that LSD1-engaged DNA replication is mechanistically linked to the loading of TopBP1-Interacting Checkpoint and Replication Regulator (TICRR) onto the pre-RC and subsequent recruitment of CDC45 during origin firing. Together, these results reveal an unexpected role for LSD1 in euchromatic origin firing and replication timing, highlighting the importance of epigenetic regulation in the activation of replication origins. As selective inhibitors of LSD1 are being exploited as potential cancer therapeutics, our study supports the importance of leveraging an appropriate level of LSD1 to curb the side effects of anti-LSD1 therapy.

MYB transcription factors play important roles in plant responses to cold stress, but the associated underlying mechanisms remain unclear. In this study, a cold-induced MYB transcription factor, DgMYB2, was isolated from chrysanthemum (Chrysanthemum morifolium Ramat). DgMYB2 was localized to the nucleus and exhibited transactivational activity. Overexpression of DgMYB2 improved cold tolerance in chrysanthemum, while cold tolerance in the antisense suppression lines decreased compared to that of the wild type. Additionally, electrophoretic mobility shift assays, chromatin immunoprecipitation, luciferase complementary imaging analysis, and dual-luciferase reporter gene detection experiments confirmed that DgMYB2 directly targets DgGPX1 and increases the activity of glutathione peroxidase to reduce the accumulation of reactive oxygen species, thereby improving cold resistance in chrysanthemum.

The bZIP transcription factor plays a very important role in abiotic stresses, e.g. drought, salt, and low-temperature stress, but the mechanism of action at low temperature is still unclear. In this study, overexpression of DgbZIP3 led to increased tolerance of chrysanthemum (Chrysanthemum morifolium Ramat.) to cold stress, whereas antisense suppression of DgbZIP3 resulted in decreased tolerance. Electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP), luciferase complementary imaging analysis (LCI), and dual-luciferase reporter gene detection (DLA) experiments indicated that DgbZIP3 directly bound to the promoter of DgPOD and activated its expression. DgbZIP2 was identified as a DgbZIP3-interacting protein using yeast two-hybrid, co-immunoprecipitation, LCI, and bimolecular fluorescence complementation assays. Overexpression of DgbZIP2 led to increased tolerance of chrysanthemum to cold stress, whereas antisense suppression of DgbZIP2 resulted in decreased tolerance. A ChIP–qPCR experiment showed that DgbZIP2 was highly enriched in the promoter of DgPOD, while DLA, EMSA, and LCI experiments further showed that DgbZIP2 could not directly regulate the expression of DgPOD. The above results show that DgbZIP3 interacts with DgbZIP2 to regulate the expression of DgPOD to promote an increase in peroxidase activity, thereby regulating the balance of reactive oxygen species and improving the tolerance of chrysanthemum to low-temperature stress.

Arbuscular mycorrhizal symbiosis (AMS) is widespread mutualistic association between plants and fungi, which plays an essential role in nutrient exchange, enhancement in plant stress resistance, development of host, and ecosystem sustainability. Previous studies have shown that plant small secreted proteins (SSPs) are involved in beneficial symbiotic interactions. However, the role of SSPs in the evolution of AMS has not been well studied yet. In this study, we performed computational analysis of SSPs in 60 plant species and identified three AMS-specific ortholog groups containing SSPs only from at least 30% of the AMS species in this study and three AMS-preferential ortholog groups containing SSPs from both AMS and non-AMS species, with AMS species containing significantly more SSPs than non-AMS species. We found that independent lineages of monocot and eudicot plants contained genes in the AMS-specific ortholog groups and had significant expansion in the AMS-preferential ortholog groups. Also, two AMS-preferential ortholog groups showed convergent changes, between monocot and eudicot species, in gene expression in response to arbuscular mycorrhizal fungus Rhizophagus irregularis. Furthermore, conserved cis-elements were identified in the promoter regions of the genes showing convergent gene expression. We found that the SSPs, and their closely related homologs, in each of three AMS-preferential ortholog groups, had some local variations in the protein structural alignment. We also identified genes co-expressed with the Populus trichocarpa SSP genes in the AMS-preferential ortholog groups. This first plant kingdom-wide analysis on SSP provides insights on plant-AMS convergent evolution with specific SSP gene expression and local diversification of protein structures.

This manuscript has been authored by UT-Battelle, LLC under Contract No. DE-AC05-00OR22725 with the U.S. Department of Energy. The United States Government retains and the publisher, by accepting the article for publication, acknowledges that the United States Government retains a non-exclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this manuscript, or allow others to do so, for United States Government purposes. The Department of Energy will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan (http://energy.gov/downloads/doe-public-access-plan).

Genital Chlamydia trachomatis (CT) is one of the most common agents of sexually transmitted infections and can cause severe disorders. This study aimed to analyse the genetic and clinical characteristics of genital CT infection among women in Guangzhou, China.From September 2020 to August 2021, a total of 8955 female patients were enrolled in this study. The presence of genital CT was detected by real-time PCR, and 273 positive samples were randomly selected for further genetic and clinical characteristics analysis.The positive rate of genital CT infection was 7.5% (670/8955), with the highest rate in women aged 21-30 years. A total of 8 genotypes were identified: DH, J, K, and recombinant genotype Ba/D. The predominant genotype was J (n = 78, 28.6%), followed by E (n = 63, 23.1%), F (n = 48, 17.6%), and D (n = 38, 13.9%). Abnormal vaginal discharge (n = 165, 61.8%), cervical columnar epithelial ectopy (n = 124, 46.4%), vaginal itching (n = 77, 28.8%), and lower abdominal pain (n = 61, 22.8%) were the predominant symptoms. Additionally, genotype G infection exhibited a significantly higher rate of abnormal vaginal discharge (P = 0.03) and genotype D infection exhibited a higher white blood cell count (P = 0.01) than the other genotypes. Phylogenetic analysis revealed a total of 20 variants with 25 mutation positions and the H2 variant in four patients was first discovered in our study.Genotypes J, E, F, and D were the major genotypes of genital CT in Guangzhou, and they manifested as abnormal vaginal discharge, cervical columnar epithelial ectopy, vaginal itching, and lower abdominal pain. The present study provides guidance for future integrated interventions to reduce the burden of genital CT infection and accelerate the development of vaccines.

Dysregulation of lipid metabolism could lead to the development of metabolic disorders. We report here that the F-box protein JFK promotes excessive lipid accumulation in adipose tissue and contributes to the development of metabolic syndrome. JFK transgenic mice develop spontaneous obesity, accompanied by dyslipidemia, hyperglycemia, and insulin resistance, phenotypes that are further exacerbated under high-fat diets. In contrast, Jfk knockout mice are lean and resistant to diet-induced metabolic malfunctions. Liver-specific reconstitution of JFK expression in Jfk knockout mice leads to hepatic lipid accumulation resembling human hepatic steatosis and nonalcoholic fatty liver disease. We show that JFK interacts with and destabilizes ING5 through assembly of the SCF complex. Integrative transcriptomic and genomic analysis reveals that the SCFJFK -ING5 axis interferes with AMPK activity and fatty acid β-oxidation, leading to the suppression of hepatic lipid catabolism. Significantly, JFK is upregulated and AMPKα1 is down-regulated in liver tissues from NAFLD patients. These results reveal that SCFJFK is a bona fide E3 ligase for ING5 and link the SCFJFK -ING5 axis to the development of obesity and metabolic syndrome.

Small secreted proteins (SSPs) are less than 250 amino acids in length and are actively transported out of cells through conventional protein secretion pathways or unconventional protein secretion pathways. In plants, SSPs have been found to play important roles in various processes, including plant growth and development, plant response to abiotic and biotic stresses, and beneficial plant–microbe interactions. Over the past 10 years, substantial progress has been made in the identification and functional characterization of SSPs in several plant species relevant to agriculture, bioenergy, and horticulture. Yet, there are potentially a lot of SSPs that have not been discovered in plant genomes, which is largely due to limitations of existing computational algorithms. Recent advances in genomics, transcriptomics, and proteomics research, as well as the development of new computational algorithms based on machine learning, provide unprecedented capabilities for genome-wide discovery of novel SSPs in plants. In this review, we summarize known SSPs and their functions in various plant species. Then we provide an update on the computational and experimental approaches that can be used to discover new SSPs. Finally, we discuss strategies for elucidating the biological functions of SSPs in plants.

Green fluorescent protein (GFP) has been widely used for monitoring gene expression and protein localization in diverse organisms. However, highly sensitive imaging equipment, like fluorescence microscope, is usually required for the visualization of GFP, limitings its application to fixed locations in samples. A reporter that can be visualized in real-time regardless the shape, size and location of the target samples will increase the flexibility and efficiency of research work. Here, we report the application of a GFP-like protein, called eYGFPuv, in both transient expression and stable transformation, in two herbaceous plant species (Arabidopsisand tobacco) and two woody plant species (poplar and citrus). We observed bright fluorescence under UV light in all of the four plant species without any effects on plant growth or development. eYGFPuv was shown to be effective for imaging transient expression in leaf and root tissues. With a focus on in vitro transformation, we demonstrated that the transgenic events expressing 1xeYGFPuvcould be easily identified visually during the callus stage and the shoot stage, enabling early and efficient selection of transformants. Furthermore, whole-plant level visualization of eYGFPuv revealed its ubiquitous stability in transgenic plants. In addition, our transformation experiments showed that eYGFPuv can also be used to select transgenic plants without antibiotics. This work demonstrates the feasibility of utilizing 1xeYGFPuvin studies of gene expression and plant transformation in diverse plants.

Plants with crassulacean acid metabolism (CAM) generally utilize water 20%–80% more efficiently than non-CAM plants. The whole genomes of several CAM plants have been sequenced or are being sequenced. For effective genome characterization and genome editing of CAM plants, an efficient transformation system is essential. In this study, we developed an Agrobacterium-mediated transformation protocol for Kalanchoe laxiflora, an obligate CAM plant,by optimizing several factors affecting the transformation efficiency. Agrobacterium strains AGL1, C58, EHA105, and GV3101 were all suitable for K. laxiflora transformation. Fifty-nine percent of the leaf explants yielded kanamycin-resistant and GUS-positive shoots. Polymerase chain reaction and quantitative real-time PCR (qRT-PCR) using gusA-, gusPlus-, nptII- and hpt-specific primers confirmed that the transgenes were integrated into K. laxiflora genome and expressed. This efficient transformation system will allow effective functional characterization of genes through over- or down-expression, knockout, or genome editing. Keywords: Kalanchoe laxiflora, Agrobacterium-mediated transformation, CAM plants

The scope and variety of the metabolic intermediates from the mitochondrial tricarboxylic acid (TCA) cycle that are engaged in epigenetic regulation of the chromatin function in the nucleus raise an outstanding question about how timely and precise supply/consumption of these metabolites is achieved in the nucleus. We report here the identification of a nonclassical TCA cycle in the nucleus (nTCA cycle). We found that all the TCA cycle-associated enzymes including citrate synthase (CS), aconitase 2 (ACO2), isocitrate dehydrogenase 3 (IDH3), oxoglutarate dehydrogenase (OGDH), succinyl-CoA synthetase (SCS), fumarate hydratase (FH), and malate dehydrogenase 2 (MDH2), except for succinate dehydrogenase (SDH), a component of electron transport chain for generating ATP, exist in the nucleus. We showed that these nuclear enzymes catalyze an incomplete TCA cycle similar to that found in cyanobacteria. We propose that the nTCA cycle is implemented mainly to generate/consume metabolic intermediates, not for energy production. We demonstrated that the nTCA cycle is intrinsically linked to chromatin dynamics and transcription regulation. Together, our study uncovers the existence of a nonclassical TCA cycle in the nucleus that links the metabolic pathway to epigenetic regulation.

A grand challenge facing society is climate change caused mainly by rising CO 2 concentration in Earth’s atmosphere. Terrestrial plants are linchpins in global carbon cycling, with a unique capability of capturing CO 2 via photosynthesis and translocating captured carbon to stems, roots, and soils for long-term storage. However, many researchers postulate that existing land plants cannot meet the ambitious requirement for CO 2 removal to mitigate climate change in the future due to low photosynthetic efficiency, limited carbon allocation for long-term storage, and low suitability for the bioeconomy. To address these limitations, there is an urgent need for genetic improvement of existing plants or construction of novel plant systems through biosystems design (or biodesign). Here, we summarize validated biological parts (e.g., protein-encoding genes and noncoding RNAs) for biological engineering of carbon dioxide removal (CDR) traits in terrestrial plants to accelerate land-based decarbonization in bioenergy plantations and agricultural settings and promote a vibrant bioeconomy. Specifically, we first summarize the framework of plant-based CDR (e.g., CO 2 capture, translocation, storage, and conversion to value-added products). Then, we highlight some representative biological parts, with experimental evidence, in this framework. Finally, we discuss challenges and strategies for the identification and curation of biological parts for CDR engineering in plants.

β-Globin gene mutations reduce or terminate the production of beta globin chains, of which approximately 10% are large deletions within the β-globin gene cluster. Because gene deletion leads to loss of heterozygosity at single nucleotide polymorphism (SNP), a novel method for detecting β-globin gene cluster deletions based on SNP heterozygosity analysis was established in this study. The location range of SNPs was selected according to the breakpoint of β-globin gene cluster deletions. SNPs were screened using bioinformatics analysis and population sequencing data. A novel method which enables genotyping of multiplex SNPs based on tetra-primer ARMS-PCR was designed and optimized. Forty clinical samples were tested in parallel by this method and MLPA to verify the performance of this method for detecting β-globin gene cluster deletion. Six informative SNPs were obtained, achieving heterozygote coverage of 93.3% in normal individuals. Genotyping of six SNPs were successfully integrated into two multiplex tetra-primer ARMS-PCR reactions. The sensitivity, specificity, positive predictive value and negative predictive value of the method for detecting β-globin gene cluster deletion were 100%, 96.30%, 92.86%, and 100%, respectively. This is a simple, cost-effective and novel method for detecting β-globin gene cluster deletions, which may be suitable for use in combination with MLPA for thalassemia molecular testing.

In January 2020, a COVID-19 outbreak was detected in Sichuan Province of China. Six weeks later, the outbreak was successfully contained. The aim of this work is to characterize the epidemiology of the Sichuan outbreak and estimate the impact of interventions in limiting SARS-CoV-2 transmission. We analyzed patient records for all laboratory-confirmed cases reported in the province for the period of January 21 to March 16, 2020. To estimate the basic and daily reproduction numbers, we used a Bayesian framework. In addition, we estimated the number of cases averted by the implemented control strategies. The outbreak resulted in 539 confirmed cases, lasted less than two months, and no further local transmission was detected after February 27. The median age of local cases was 8 years older than that of imported cases. We estimated R0 at 2.4 (95% CI: 1.6–3.7). The epidemic was self-sustained for about 3 weeks before going below the epidemic threshold 3 days after the declaration of a public health emergency by Sichuan authorities. Our findings indicate that, were the control measures be adopted four weeks later, the epidemic could have lasted 49 days longer (95% CI: 31–68 days), causing 9,216 more cases (95% CI: 1,317–25,545)., Author summary Since its emergence in Wuhan, SARS-CoV-2 rapidly started its spread across China. On January 21, 2020 the first COVID-19 case was detected in the Sichuan Province of China and led to an outbreak of local transmission. Less than two months later, the outbreak was over with the last reported case on March 4, 2020. In this study, we analyzed patient records for all laboratory-confirmed cases reported in Sichuan to provide an epidemiological characterization of the outbreak, to estimate SARS-CoV-2 transmission potential, and to assess the impact of the adopted interventions. We estimated that, during the initial exponential growth phase of the outbreak, each COVID-19 case has generated a mean of 2.4 secondary cases (95% CI: 1.6–3.7). Moreover, we estimated that, were the Sichuan strict containment measures implemented four weeks later, the outbreak would have caused 9,216 more cases (95% CI: 1,317–25,545). Our findings suggest the key role of a quick response to COVID-19 outbreaks and the importance of an adequate surveillance and monitoring system.

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 C 3 or C 4 photosynthesis. CAM plants are derived from C 3 photosynthesis ancestors. However, it is extremely unlikely that the C 3 or C 4 crop plants would evolve rapidly into CAM photosynthesis without human intervention. Currently, there is growing interest in improving WUE through transferring CAM into C 3 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 C 3 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 C 3 -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 C 3 -to-CAM transition in plants using synthetic biology toolboxes.

The long atmospheric residence time of CO 2 creates an urgent need to add atmospheric carbon drawdown to CO 2 regulatory strategies. Synthetic and systems biology (SSB), which enables manipulation of cellular phenotypes, offers a powerful approach to amplifying and adding new possibilities to current land management practices aimed at reducing atmospheric carbon. The participants (in attendance: Christina Agapakis, George Annas, Adam Arkin, George Church, Robert Cook-Deegan, Charles DeLisi, Dan Drell, Sheldon Glashow, Steve Hamburg, Henry Jacoby, Henry Kelly, Mark Kon, Todd Kuiken, Mary Lidstrom, Mike MacCracken, June Medford, Jerry Melillo, Ron Milo, Pilar Ossorio, Ari Patrinos, Keith Paustian, Kristala Jones Prather, Kent Redford, David Resnik, John Reilly, Richard J. Roberts, Daniel Segre, Susan Solomon, Elizabeth Strychalski, Chris Voigt, Dominic Woolf, Stan Wullschleger, and Xiaohan Yang) identified a range of possibilities by which SSB might help reduce greenhouse gas concentrations and which might also contribute to environmental sustainability and adaptation. These include, among other possibilities, engineering plants to convert CO 2 produced by respiration into a stable carbonate, designing plants with an increased root-to-shoot ratio, and creating plants with the ability to self-fertilize. A number of serious ecological and societal challenges must, however, be confronted and resolved before any such application can be fully assessed, realized, and deployed.

Many applications in plant biology requires editing genomes accurately including correcting point mutations, incorporation of single-nucleotide polymorphisms (SNPs), and introduction of multinucleotide insertion/deletions (indels) into a predetermined position in the genome. These types of modifications are possible using existing genome-editing technologies such as the CRISPR-Cas systems, which require induction of double-stranded breaks in the target DNA site and the supply of a donor DNA molecule that contains the desired edit sequence. However, low frequency of homologous recombination in plants and difficulty of delivering the donor DNA molecules make this process extremely inefficient. Another kind of technology known as base editing can perform precise editing; however, only certain types of modifications can be obtained, e.g., C/G-to-T/A and A/T-to-G/C. Recently, a new type of genome-editing technology, referred to as “prime editing,” has been developed, which can achieve various types of editing such as any base-to-base conversion, including both transitions (C→T, G→A, A→G, and T→C) and transversion mutations (C→A, C→G, G→C, G→T, A→C, A→T, T→A, and T→G), as well as small indels without the requirement for inducing double-stranded break in the DNA. Because prime editing has wide flexibility to achieve different types of edits in the genome, it holds a great potential for developing superior crops for various purposes, such as increasing yield, providing resistance to various abiotic and biotic stresses, and improving quality of plant product. In this review, we describe the prime editing technology and discuss its limitations and potential applications in plant biology research.

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.

For decades, plants have been the subject of genetic engineering to synthesize novel, value-added compounds. Polyhydroxyalkanoates (PHAs), a large class of biodegradable biopolymers naturally synthesized in eubacteria, are among the novel products that have been introduced to make use of plant acetyl-CoA metabolic pathways. It was hoped that renewable PHA production would help address environmental issues associated with the accumulation of nondegradable plastic wastes. However, after three decades of effort synthesizing PHAs, and in particular the simplest form polyhydroxybutyrate (PHB), and seeking to improve their production in plants, it has proven very difficult to reach a commercially profitable rate in a normally growing plant. This seems to be due to the growth defects associated with PHA production and accumulation in plant cells. Here, we review major breakthroughs that have been made in plant-based PHA synthesis using traditional genetic engineering approaches and discuss challenges that have been encountered. Then, from the point of view of plant synthetic biology, we provide perspectives on reprograming plant acetyl-CoA pathways for PHA production, with the goal of maximizing PHA yield while minimizing growth inhibition. Specifically, we suggest genetic elements that can be considered in genetic circuit design, approaches for nuclear genome and plastome modification, and the use of multiomics and mathematical modeling in understanding and restructuring plant metabolic pathways.

Coxsackievirus A6 (CA6) infection may lead to high hand-foot-and-mouth disease (HFMD) aggregation in children. We aimed to analyze the clinical and phylogenetic features of severe CA6-associated pediatric HFMD.The clinical and laboratory features of 206 and 55 children with mild and severe CA6-associated HFMD, respectively, were summarized. The CA6 phylogenetic tree was depicted using combinatorial analysis of the VP1-encoding regions and neighbor-joining method.CA6 was the major pathogen both in mild and severe HFMD in 2017. Most CA6-associated severe HFMD cases showed high fever, skin rash, age younger than 36 months, and elevated white blood cell and C-reactive protein levels, and there were no significant differences compared to the mild cases (p 0.05). The severe cases were significantly more likely (p 0.05) to show male sex, long fever duration, decreased oral intake, tonsil enlargement, diarrhea, vomiting, elevated levels of creatine kinase and blood glucose, and positive fecal occult-blood test results. Severe complications included aseptic meningitis (29/55, 52.7%) and pulmonary edema (6/55, 10.9%) were observed in severe cases. Furthermore, genetic analyses showed all CA6 isolates belonged to lineage E2, and two amino acid changes of V174I and T283A in VP1 may be associated with the severity of HFMD.CA6 has become a major cause of HFMD with severe systemic disorders. V174I and T283A of VP1 may be associated with the severity of CA6 infection. These findings could raise awareness of the clinical importance of CA6 infection among practitioners.

Author(s): Yin, Hengfu; Guo, Hao-Bo; Weston, David J; Borland, Anne M; Ranjan, Priya; Abraham, Paul E; Jawdy, Sara S; Wachira, James; Tuskan, Gerald A; Tschaplinski, Timothy J; Wullschleger, Stan D; Guo, Hong; Hettich, Robert L; Gross, Stephen M; Wang, Zhong; Visel, Axel; Yang, Xiaohan | Abstract: Following publication of the original article.

Ecto- and endo-mycorrhizal colonization of Populus roots have a positive impact on the overall tree health and growth.[br/] A complete molecular understanding of these interactions will have important implications for increasing agricultural or forestry sustainability using plant: microbe-based strategies. These beneficial associations entail extensive morphological changes orchestrated by the genetic reprogramming in both organisms.[br/] In this study, we performed a comparative analysis of two Populus species (Populus deltoides and P. trichocarpa) that were colonized by either an arbuscular mycorrhizal fungus (AmF), Rhizophagus irregularis or an ectomycorrhizal fungus (EmF), Laccaria bicolor, to describe the small RNA (sRNA) landscape including small open reading frames (sORFs) and micro RNAs (miRNAs) involved in these mutualistic interactions.[br/] We identified differential expression of sRNAs that were, to a large extent, (1) within the genomic regions lacking annotated genes in the Populus genome and (2) distinct for each fungal interaction. These sRNAs may be a source of novel sORFs within a genome, and in this regard, we identified potential sORFs encoded by the sRNAs. We predicted a higher number of differentially-expressed miRNAs in P. trichocarpa (4 times more) than in P. deltoides (conserved and novel). In addition, 44 miRNAs were common in P. trichocarpa between the EmF and AmF treatments, and only 4 miRNAs were common in P. deltoides between the treatments. Root colonization by either fungus was more effective in P. trichocarpa than in P. deltoides, thus the relatively few differentiallyexpressed miRNAs predicted in P. deltoides might reflect the extent of the symbiosis. Finally, we predicted several genes targets for the plant miRNAs identified here, including potential fungal gene targets.[br/] Our findings shed light on additional molecular tiers with a role in Populus-fungal mutualistic associations and provides a set of potential molecular targets for future enhancement.

Genome-wide association studies (GWAS) have great promise for identifying the loci that contribute to adaptive variation, but the complex genetic architecture of many quantitative traits presents a substantial challenge. We measured 14 morphological and physiological traits and identified single nucleotide polymorphism (SNP)-phenotype associations in a Populus trichocarpa population distributed from California, USA to British Columbia, Canada. We used whole-genome resequencing data of 882 trees with more than 6.78 million SNPs, coupled with multitrait association to detect polymorphisms with potentially pleiotropic effects. Candidate genes were validated with functional data. Broad-sense heritability (H2 ) ranged from 0.30 to 0.56 for morphological traits and 0.08 to 0.36 for physiological traits. In total, 4 and 20 gene models were detected using the single-trait and multitrait association methods, respectively. Several of these associations were corroborated by additional lines of evidence, including co-expression networks, metabolite analyses, and direct confirmation of gene function through RNAi. Multitrait association identified many more significant associations than single-trait association, potentially revealing pleiotropic effects of individual genes. This approach can be particularly useful for challenging physiological traits such as water-use efficiency or complex traits such as leaf morphology, for which we were able to identify credible candidate genes by combining multitrait association with gene co-expression and co-methylation data.

Background Crassulacean acid metabolism (CAM) enhances plant water-use efficiency through an inverse day/night pattern of stomatal closure/opening that facilitates nocturnal CO2 uptake. CAM has evolved independently in over 35 plant lineages, accounting for ~ 6% of all higher plants. Agave species are highly heat- and drought-tolerant, and have been domesticated as model CAM crops for beverage, fiber, and biofuel production in semi-arid and arid regions. However, the genomic basis of evolutionary innovation of CAM in genus Agave is largely unknown. Results Using an approach that integrated genomics, gene co-expression networks, comparative genomics and protein structure analyses, we investigated the molecular evolution of CAM as exemplified in Agave. Comparative genomics analyses among C3, C4 and CAM species revealed that core metabolic components required for CAM have ancient genomic origins traceable to non-vascular plants while regulatory proteins required for diel re-programming of metabolism have a more recent origin shared among C3, C4 and CAM species. We showed that accelerated evolution of key functional domains in proteins responsible for primary metabolism and signaling, together with a diel re-programming of the transcription of genes involved in carbon fixation, carbohydrate processing, redox homeostasis, and circadian control is required for the evolution of CAM in Agave. Furthermore, we highlighted the potential candidates contributing to the adaptation of CAM functional modules. Conclusions This work provides evidence of adaptive evolution of CAM related pathways. We showed that the core metabolic components required for CAM are shared by non-vascular plants, but regulatory proteins involved in re-reprogramming of carbon fixation and metabolite transportation appeared more recently. We propose that the accelerated evolution of key proteins together with a diel re-programming of gene expression were required for CAM evolution from C3 ancestors in Agave. Electronic supplementary material The online version of this article (10.1186/s12864-018-4964-7) contains supplementary material, which is available to authorized users.

Drought stress is a recurring feature of world climate and the single most important factor influencing agricultural yield worldwide. Plants display highly variable, species-specific responses to drought and these responses are multifaceted, requiring physiological and morphological changes influenced by genetic and molecular mechanisms. Moreover, the reproducibility of water deficit studies is very cumbersome, which significantly impedes research on drought tolerance, because how a plant responds is highly influenced by the timing, duration, and intensity of the water deficit. Despite progress in the identification of drought-related mechanisms in many plants, the molecular basis of drought resistance remains to be fully understood in trees, particularly in poplar species because their wide geographic distribution results in varying tolerances to drought. Herein, we aimed to better understand this complex phenomenon in eastern cottonwood (Populus deltoides) by performing a detailed contrast of the proteome changes between two different water deficit experiments to identify functional intersections and divergences in proteome responses. We investigated plants subjected to cyclic water deficit and compared these responses to plants subjected to prolonged acute water deficit. In total, we identified 108,012 peptide sequences across both experiments that provided insight into the quantitative state of 22,737 Populus gene models and 8,199 functional protein groups in response to drought. Together, these datasets provide the most comprehensive insight into proteome drought responses in poplar to date and a direct proteome comparison between short period dehydration shock and cyclic, post-drought re-watering. Overall, this investigation provides novel insights into drought avoidance mechanisms that are distinct from progressive drought stress. Additionally, we identified proteins that have been associated as drought-relevant in previous studies. Importantly, we highlight the RD26 transcription factor as a gene regulated at both the transcript and protein level, regardless of species and drought condition, and, thus, represents a key, universal drought marker for Populus species.

Due to public concerns about the decreasing supply of blue water and increasing heat and drought stress on plant growth caused by urbanization, increasing human population and climate change, interest in crassulacean acid metabolism (CAM), a specialized type of photosynthesis enhancing water-use efficiency (WUE) and drought tolerance, has increased markedly. Significant progress has been achieved in both basic and applied research in CAM plants since the beginning of this century. Here we provide a brief overview of the current status of CAM research, and discuss future needs and opportunities in a wide range of areas including systems biology, synthetic biology, and utilization of CAM crops for human benefit, with a focus on the following aspects: 1) application of genome-editing technology and high-throughput phenotyping to functional genomics research in model CAM species and genetic improvement of CAM crops, 2) challenges for multi-scale metabolic modeling of CAM systems, 3) opportunities and new strategies for CAM pathway engineering to enhance WUE and drought tolerance in C3 (and C4) photosynthesis crops, 4) potential of CAM species as resources for food, feed, natural products, pharmaceuticals and biofuels, and 5) development of CAM crops for ecological and aesthetic benefits.

Crassulacean acid metabolism (CAM) is a water-use efficient adaptation of photosynthesis that has evolved independently many times in diverse lineages of flowering plants. We hypothesize that convergent evolution of protein sequence and temporal gene expression underpins the independent emergences of CAM from C3 photosynthesis. To test this hypothesis, we generate a de novo genome assembly and genome-wide transcript expression data for Kalanchoë fedtschenkoi, an obligate CAM species within the core eudicots with a relatively small genome (~260 Mb). Our comparative analyses identify signatures of convergence in protein sequence and re-scheduling of diel transcript expression of genes involved in nocturnal CO2 fixation, stomatal movement, heat tolerance, circadian clock, and carbohydrate metabolism in K. fedtschenkoi and other CAM species in comparison with non-CAM species. These findings provide new insights into molecular convergence and building blocks of CAM and will facilitate CAM-into-C3 photosynthesis engineering to enhance water-use efficiency in crops., Crassulacean acid metabolism (CAM) is a metabolic adaptation of photosynthesis that enhances water use efficiency. Here, via genomic analysis of Kalanchoë, the authors provide evidence for convergent evolution of protein sequence and temporal gene expression underpinning the multiple independent emergences of CAM.

The existence of complete genome sequences makes it important to develop different approaches for classification of large-scale data sets and to make extraction of biological insights easier. Here, we propose an approach for classification of complete proteomes/protein sets based on protein distributions on some basic attributes. We demonstrate the usefulness of this approach by determining protein distributions in terms of two attributes: protein lengths and protein intrinsic disorder contents (ID). The protein distributions based on L and ID are surveyed for representative proteome organisms and protein sets from the three domains of life. The two-dimensional maps (designated as fingerprints here) from the protein distribution densities in the LD space defined by ln(L) and ID are then constructed. The fingerprints for different organisms and protein sets are found to be distinct with each other, and they can therefore be used for comparative studies. As a test case, phylogenetic trees have been constructed based on the protein distribution densities in the fingerprints of proteomes of organisms without performing any protein sequence comparison and alignments. The phylogenetic trees generated are biologically meaningful, demonstrating that the protein distributions in the LD space may serve as unique phylogenetic signals of the organisms at the proteome level.

Non-coding RNAs (ncRNAs), that is, RNAs not translated into proteins, are crucial regulators of a variety of biological processes in plants. While protein-encoding genes have been relatively well-annotated in sequenced genomes, accounting for a small portion of the genome space in plants, the universe of plant ncRNAs is rapidly expanding. Recent advances in experimental and computational technologies have generated a great momentum for discovery and functional characterization of ncRNAs. Here we summarize the classification and known biological functions of plant ncRNAs, review the application of next-generation sequencing (NGS) technology and ribosome profiling technology to ncRNA discovery in horticultural plants and discuss the application of new technologies, especially the new genome-editing tool clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) systems, to functional characterization of plant ncRNAs.