CRISPR/Cas9-based gene activation and base editing in Populus

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”(P.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 hybrid 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.

Convolutional Neural Network Based on Spatial Pyramid for Image Classification

A novel convolutional neural network based on spatial pyramid for image classification is proposed.The network exploits image features with spatial pyramid representation.First,it extracts global features from an original image,and then different layers of grids are utilized to extract feature maps from different convolutional layers.Inspired by the spatial pyramid,the new network contains two parts,one of which is just like a standard convolutional neural network,composing of alternating convolutions and subsampling layers.But those convolution layers would be averagely pooled by the grid way to obtain feature maps,and then concatenated into a feature vector individually.Finally,those vectors are sequentially concatenated into a total feature vector as the last feature to the fully connection layer.This generated feature vector derives benefits from the classic and previous convolution layer,while the size of the grid adjusting the weight of the feature maps improves the recognition efficiency of the network.Experimental results demonstrate that this model improves the accuracy and applicability compared with the traditional model.

Expanding the application of a UV-visible reporter for transient gene expression and stable transformation in plants

Green fl uorescent protein(GFP)has been widely used for monitoring gene expression and protein localization in diverse organisms.However,highly sensitive imaging equipment,like fl uorescence microscope,is usually required for the visualization of GFP,limitings its application to fi xed locations in samples.A reporter that can be visualized in realtime regardless the shape,size and location of the target samples will increase the fl exibility and ef fi ciency 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(Arabidopsis and tobacco)and two woody plant species(poplar and citrus).We observed bright fl uorescence 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 1x eYGFPuv could be easily identi fi ed visually during the callus stage and the shoot stage,enabling early and ef fi cient 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 1x eYGFPuv in studies of gene expression and plant transformation in diverse plants.

Advances and perspectives in discovery and functional analysis of small secreted proteins in plants

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.

Neutron yield measurement system of HL-2A tokamak

This research presents the development of HL-2A neutron yield measurement which includes^(235)U fission chamber and BF_(3)and^(3)He proportional counters.Equivalent noise formula of the radiation detection signal amplification system was derived to guide the development of the signal amplification system.Then all detectors were calibrated in situ by using the^(252)C_(f)neutron source.The neutron yield of the HL-2A during neutral beam heating was analyzed.These results indicate that the developed neutron flux diagnostic system can obtain neutron yield results under various experimental conditions of the HL-2A tokamak,and can provide information on neutron yield.

A review of flexible perovskite oxide ferroelectric films and their application

Many perovskite oxide ferroelectrics(e.g.PbZr_(1-x)Ti_(x)O_(3),BaTiO_(3),LiNbO_(3))are born with multitudinous robust performances and have been widely used in sensors,actuators,surface acoustic wave devices,and memories et al.However,their hardness,brittleness and harsh synthesis conditions(i.e.high temperature and oxygen ambience)restrain their application into flexible electronic devices which are significant components among the three pillars of modern society development,i.e.energy,information and materials.Here we review the preparation of flexible devices based on these oxide ferroelectrics,including transferring these freestanding films to flexible substrates after separating ferroelectric oxide films from the hard substrates,such as Si and SrTiO_(3) crystals,and also direct fabrication methods without transferring process.Subsequently,we summarize three kinds of representative flexible devices,i.e.flexible ferroelectric memories,sensors and generators.These inorganic electronics not only show excellent electric properties competitive with those corresponding electronics on hard substrates but also exhibit good flexibility similar to many organic flexible electronics.

Flexible memristors as electronic synapses for neuro- inspired computation based on scotch tape-exfoliated mica substrates

Flexible memristor devices based on plastic substrates have attracted considerable attention due to their applications in wearable computers and integrated circuits. However, most plastic-substrate memristors cannot function or be grown in high-temperature environments. In this study, scotch-tape-exfoliated mica was used as the flexible memristor substrate in order to resolve these high-temperature issues. Our TiN/ZHO/IGZO memristor, which was constructed using a thin （10 μm） mica substrate, has superior flexibility and thermostability. After bending it 103 times, the device continues to exhibit exceptional electrical characteristics. It can also be implemented for transitions between high and low resistance states, even in temperatures of up to 300 ℃. More importantly, the biological synaptic characteristics of paired-pulse facilitation/depression （PPF/PPD） and spike- timing-dependent plasticity （STDP） were observed through applying different pulse measurement modes. This work demonstrates that flexible memristor devices on mica substrates may potentially allow for the realization of high-temperature memristor applications for biologically-inspired computing systems.

Toward artificial intelligent self-cooling electronic skins:Large electrocaloric effect in all-inorganic flexible thin films at room temperature

Intelligent robots have assisted mankind in achieving and operating thousands of functions,especially with the arrival of the artificial intelligent.However,heat dissipation and thermal management in the intelligent robots remain big challenges,which limit their miniaturization and performance.Electrocaloric(EC)materials,which exhibit temperature change in response to the application or withdrawal of an electric field,open a new strategy for cooling technology and have gained a flurry of research interest in recent years.Toward artificial intelligent self-cooling electronic skins,large-scale flexible materials with high EC effect near room temperature are in demand.Here,we report a large room temperature EC effect in flexible Pb_(0.82)Ba_(0.08)La_(0.1)Zr0.9Ti_(0.1)O_(3)(PBLZT)inorganic thin films via a transfer-free cost-effective sol-gel process,assisted by unique two-dimensional mica substrates.The maximum adiabatic temperature change and isothermal entropy change of the flexible PBLZT thin films reach to 22.5 K and 25.9 J K^(-1) kg^(-1) at room temperature.In particular,the flexible PBLZT thin films exhibit a stable EC effect both under bending state and after bending for 20000 times.Our flexible EC materials offer an alternative strategy to the development of cooling technologies for both artificial intelligent robots and personal wearable cooling devices.

Bio-inspired flexible vibration visualization sensor based on piezo-electrochromic effect

Monitoring structural vibration can provide quantitative information for both structural health evaluations and early-warning maintenance.The most classic vibration-based structural health monitoring is equipped with piezoelectric accelerometers,which is expensive and inconvenient due to cumbersome and time-consuming sensor installation and high power-consumptive data acquisition systems.One other main challenge with these systems is the inherent limitations for multi-point monitoring in critical elements with curvature due to their non-conformability.Here,inspired by the chameleon,we report a cost-effective,flexible and conformal,self-powered vibration sensor based on elasto-electro-chemical synergistic effect of piezoelectricity and electrochromism.The sensor can provide not only in-situ visualization,but also ex-situ recording of structural vibration due to the non-volatile color memory effect of electrochromism.The passive sensor system is composed of two distinct electronic components d ternary Pb(In1/2Nb1/2)O_(3)ePb(Mg1/3Nb2/3)O_(3)ePbTiO_(3)piezoelectric single crystal ribbon sensors and a solid-state tungsten trioxide electrochromic indicator driven by vibration-induced voltage generated by the piezoelectric ribbons.The proposed piezo-electrochromic based passive non-volatile visualization sensor may find diverse applications in structural health monitoring,smart wallpapers,and medical injury rehabilitation.

External stimuli controlled multiferroic charge-transfer crystals

Multiferroic charge-transfer crystals have drawn significant interest due to their simultaneous dipolar and spin ordering. Numerous theoretical and experimental studies have shown that the molecular stacking between donor and acceptor complexes plays an important role in tuning charge-transfer enabled multifunctionality. Herein, we show that the charge-transfer interactions can be controlled by the segregated stack, consisting of polythiophene donor- and fuUerene acceptor-based all-conjugated block copolymers. Room temperature magnetic field effects, ferroelectricity, and anisotropic magnetism are observed in charge-transfer crystals, which can be further controlled by photoexcitation and charge doping. Furthermore, the charge-transfer segregated stack crystals demonstrate external stimuli controlled polarization and magnetization, which opens up their multifunctional applications for all-organic multiferroics.

The development of Bi Fe O_3-based ceramics

The multiferroic properties of Bi Fe O3-based ceramics were improved through optimizing their sintering method and doping with certain rare earth elements in pure Bi Fe O3. Some methods, especially liquid-phase sintering method has largely decreased the densities of oxygen vacancies and Fe2in Bi Fe O3-based ceramics, and thus their resistivity became high enough to measure the saturated polarization and the large piezoelectric d33 coefficient under the high electric field of [150 k V/cm. Besides,multiferroic properties were improved through the rare earth elements' doping in pure Bi Fe O3. Magnetization commonly increases with the proportional increase of Nd,La, Sm and Dy contents up to *30 %, while ferroelectric phase can transform to paraelectric phase at a certain proportion. An improved magnetoelectric coupling was often observed at ferroelectric phase with a relatively large proportion. Besides, an enhanced piezoelectric coefficient is expected in Bi Fe O3-based ceramics with morphotropic phase boundaries as they are already observed in thin epitaxial Bi Fe O3 films.

Plant Biosystems Design Research Roadmap 1.0

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

Biosystems Design to Accelerate C3-to-CAM Progression

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

Reconfiguring Plant Metabolism for Biodegradable Plastic Production

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.

Prime Editing Technology and Its Prospects for Future Applications in Plant Biology Research

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.

Biological and Molecular Components for Genetically Engineering Biosensors in Plants

Plants adapt to their changing environments by sensing and responding to physical,biological,and chemical stimuli.Due to their sessile lifestyles,plants experience a vast array of external stimuli and selectively perceive and respond to specific signals.By repurposing the logic circuitry and biological and molecular components used by plants in nature,genetically encoded plant-based biosensors(GEPBs)have been developed by directing signal recognition mechanisms into carefully assembled outcomes that are easily detected.GEPBs allow for in vivo monitoring of biological processes in plants to facilitate basic studies of plant growth and development.GEPBs are also useful for environmental monitoring,plant abiotic and biotic stress management,and accelerating design-build-test-learn cycles of plant bioengineering.With the advent of synthetic biology,biological and molecular components derived from alternate natural organisms(e.g.,microbes)and/or de novo parts have been used to build GEPBs.In this review,we summarize the framework for engineering different types of GEPBs.We then highlight representative validated biological components for building plant-based biosensors,along with various applications of plant-based biosensors in basic and applied plant science research.Finally,we discuss challenges and strategies for the identification and design of biological components for plant-based biosensors.

Biological Parts for Plant Biodesign to Enhance Land-Based Carbon Dioxide Removal

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.

Plant Promoters and Terminators for High-Precision Bioengineering

High-precision bioengineering and synthetic biology require fine-tuning gene expression at both transcriptional and posttranscriptional levels.Gene transcription is tightly regulated by promoters and terminators.Promoters determine the timing,tissues and cells,and levels of the expression of genes.Terminators mediate transcription termination of genes and affect mRNA levels posttranscriptionally,e.g.,the 3′-end processing,stability,translation efficiency,and nuclear to cytoplasmic export of mRNAs.The promoter and terminator combination affects gene expression.In the present article,we review the function and features of plant core promoters,proximal and distal promoters,and terminators,and their effects on and benchmarking strategies for regulating gene expression.