Heritable, multinucleotide deletions in plants using viral delivery of a repair exonuclease and guide RNAs.

CRISPR/Cas9-mediated mutagenesis typically results in short insertion/deletion mutations, which are often too small to disrupt the function of cis-acting regulatory elements. Here, we describe a highly efficient in planta gene editing approach called VirTREX2-HLDel that achieves heritable multinucleotide deletions in both protein-coding genes and noncoding DNA regulatory elements. VirTREX2-HLDel uses RNA viruses to deliver both the 3 prime repair exonuclease 2 (TREX2) and single-guide RNAs. Our method enables recovery of multiplexed heritable deletions and increases the heritable gene editing frequency at poorly edited sites. We identified functional conservation and divergence of MICRORNA164 (miR164) in Nicotiana benthamiana and tomato (Solanum lycopersicum) using VirTREX2-HLDel and observed previously uncharacterized phenotypes in plants with large deletions at this locus. Our viral delivery method reduces the need for tissue culture and will accelerate the understanding of protein-coding and regulatory regions in plants.

Elucidating the role of Brain-Derived Neurotrophic Factor (BDNF) and its receptor Tyrosine Receptor Kinase B (TrkB) in the development and symptoms of endometriosis.

Endometriosis (EMs) is a common disease among women of reproductive age, and as of now, the clinical understanding of the etiology of this disease remains unclear. The occurrence of EMs has a profound impact on the reproductive health of women, making early diagnosis and treatment of this disease a pressing challenge in clinical practice. Recent studies have found that Brain-Derived Neurotrophic Factor (BDNF), in combination with its high-affinity receptor Tyrosine Receptor Kinase B (TrkB), participates in the development of EMs and the appearance of clinically relevant symptoms by activating the Mitogen-Activated Protein Kinase (MAPK) pathway, the Phosphatidylinositol 3-Kinase/Protein Kinase B (PI3K/AKT) pathway, and the Phospholipase C-gamma (PLCγ) signaling pathway, or by interacting with other factors. In order to gain a deeper understanding of the pathogenesis related to EMs, this article reviews the roles of BDNF and TrkB in EMs, particularly in terms of aberrant apoptosis and autophagy, cell invasion, proliferation, angiogenesis, oxidative stress, and inflammatory reactions, as well as their relationship with the symptoms associated with EMs.

Agave REVEILLE1 regulates the onset and release of seasonal dormancy in Populus.

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.

Comparative genomics analysis of drought response between obligate CAM and C3 photosynthesis plants.

Crassulacean acid metabolism (CAM) plants exhibit elevated drought and heat tolerance compared to C3 and C4 plants through an inverted pattern of day/night stomatal closure and opening for CO2 assimilation. However, the molecular responses to water-deficit conditions remain unclear in obligate CAM species. In this study, we presented genome-wide transcription sequencing analysis using leaf samples of an obligate CAM species Kalanchoë fedtschenkoi under moderate and severe drought treatments at two-time points of dawn (2-h before the start of light period) and dusk (2-h before the dark period). Differentially expressed genes were identified in response to environmental drought stress and a whole genome wide co-expression network was created as well. We found that the expression of CAM-related genes was not regulated by drought stimuli in K. fedtschenkoi. Our comparative analysis revealed that CAM species (K. fedtschenkoi) and C3 species (Arabidopsis thaliana, Populus deltoides 'WV94') share some common transcriptional changes in genes involved in multiple biological processes in response to drought stress, including ABA signaling and biosynthesis of secondary metabolites.

Overexpression of an Agave Phosphoenolpyruvate Carboxylase Improves Plant Growth and Stress Tolerance.

It has been challenging to simultaneously improve photosynthesis and stress tolerance in plants. Crassulacean acid metabolism (CAM) is a CO2-concentrating mechanism that facilitates plant adaptation to water-limited environments. We hypothesized that the ectopic expression of a CAM-specific phosphoenolpyruvate carboxylase (PEPC), an enzyme that catalyzes primary CO2 fixation in CAM plants, would enhance both photosynthesis and abiotic stress tolerance. To test this hypothesis, we engineered a CAM-specific PEPC gene (named AaPEPC1) from Agave americana into tobacco. In comparison with wild-type and empty vector controls, transgenic tobacco plants constitutively expressing AaPEPC1 showed a higher photosynthetic rate and biomass production under normal conditions, along with significant carbon metabolism changes in malate accumulation, the carbon isotope ratio δ13C, and the expression of multiple orthologs of CAM-related genes. Furthermore, AaPEPC1 overexpression enhanced proline biosynthesis, and improved salt and drought tolerance in the transgenic plants. Under salt and drought stress conditions, the dry weight of transgenic tobacco plants overexpressing AaPEPC1 was increased by up to 81.8% and 37.2%, respectively, in comparison with wild-type plants. Our findings open a new door to the simultaneous improvement of photosynthesis and stress tolerance in plants.

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

A grand challenge facing society is climate change caused mainly by rising CO2 concentration in Earth's atmosphere. Terrestrial plants are linchpins in global carbon cycling, with a unique capability of capturing CO2 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 CO2 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., CO2 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.

Light-responsive expression atlas reveals the effects of light quality and intensity in Kalanchoë fedtschenkoi, a plant with crassulacean acid metabolism.

BACKGROUND: Crassulacean acid metabolism (CAM), a specialized mode of photosynthesis, enables plant adaptation to water-limited environments and improves photosynthetic efficiency via an inorganic carbon-concentrating mechanism. Kalanchoë fedtschenkoi is an obligate CAM model featuring a relatively small genome and easy stable transformation. However, the molecular responses to light quality and intensity in CAM plants remain understudied. RESULTS: Here we present a genome-wide expression atlas of K. fedtschenkoi plants grown under 12 h/12 h photoperiod with different light quality (blue, red, far-red, white light) and intensity (0, 150, 440, and 1,000 µmol m-2 s-1) based on RNA sequencing performed for mature leaf samples collected at dawn (2 h before the light period) and dusk (2 h before the dark period). An eFP web browser was created for easy access of the gene expression data. Based on the expression atlas, we constructed a light-responsive co-expression network to reveal the potential regulatory relationships in K. fedtschenkoi. Measurements of leaf titratable acidity, soluble sugar, and starch turnover provided metabolic indicators of the magnitude of CAM under the different light treatments and were used to provide biological context for the expression dataset. Furthermore, CAM-related subnetworks were highlighted to showcase genes relevant to CAM pathway, circadian clock, and stomatal movement. In comparison with white light, monochrome blue/red/far-red light treatments repressed the expression of several CAM-related genes at dusk, along with a major reduction in acid accumulation. Increasing light intensity from an intermediate level (440 µmol m-2 s-1) of white light to a high light treatment (1,000 µmol m-2 s-1) increased expression of several genes involved in dark CO2 fixation and malate transport at dawn, along with an increase in organic acid accumulation. CONCLUSIONS: This study provides a useful genomics resource for investigating the molecular mechanism underlying the light regulation of physiology and metabolism in CAM plants. Our results support the hypothesis that both light intensity and light quality can modulate the CAM pathway through regulation of CAM-related genes in K. fedtschenkoi.

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.

Comparative genomics can provide new insights into the evolutionary mechanisms and gene function in CAM plants.

Crassulacean acid metabolism (CAM) photosynthesis is an important biological innovation enabling plant adaptation to hot and dry environments. CAM plants feature high water-use efficiency, with potential for sustainable crop production under water-limited conditions. A deep understanding of CAM-related gene function and molecular evolution of CAM plants is critical for exploiting the potential of engineering CAM into C3 crops to enhance crop production on semi-arid or marginal agricultural lands. With the newly emerging genomics resources for multiple CAM species, progress has been made in comparative genomics studies on the molecular basis and subsequently on the evolution of CAM. Here, recent advances in CAM comparative genomics research in constitutive and facultative CAM plants are reviewed, with a focus on the analyses of DNA/protein sequences and gene expression to provide new insights into the path and driving force of CAM evolution and to identify candidate genes involved in CAM-related biological processes. Potential applications of new computational and experimental technologies (e.g. CRISPR/Cas-mediated genome-editing technology) to the comparative and evolutionary genomics research on CAM plants are offered.

CRISPR/Cas9-mediated targeted mutagenesis for functional genomics research of crassulacean acid metabolism plants.

Crassulacean acid metabolism (CAM) is an important photosynthetic pathway in diverse lineages of plants featuring high water-use efficiency and drought tolerance. A big challenge facing the CAM research community is to understand the function of the annotated genes in CAM plant genomes. Recently, a new genome editing technology using CRISPR/Cas9 has become a more precise and powerful tool than traditional approaches for functional genomics research in C3 and C4 plants. In this study, we explore the potential of CRISPR/Cas9 to characterize the function of CAM-related genes in the model CAM species Kalanchoë fedtschenkoi. We demonstrate that CRISPR/Cas9 is effective in creating biallelic indel mutagenesis to reveal previously unknown roles of blue light receptor phototropin 2 (KfePHOT2) in the CAM pathway. Knocking out KfePHOT2 reduced stomatal conductance and CO2 fixation in late afternoon and increased stomatal conductance and CO2 fixation during the night, indicating that blue light signaling plays an important role in the CAM pathway. Lastly, we provide a genome-wide guide RNA database targeting 45 183 protein-coding transcripts annotated in the K. fedtschenkoi genome.

Perspectives on the basic and applied aspects of crassulacean acid metabolism (CAM) research.

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.

A Vitis vinifera basic helix-loop-helix transcription factor enhances plant cell size, vegetative biomass and reproductive yield.

Strategies for improving plant size are critical targets for plant biotechnology to increase vegetative biomass or reproductive yield. To improve biomass production, a codon-optimized helix-loop-helix transcription factor (VvCEB1opt ) from wine grape was overexpressed in Arabidopsis thaliana resulting in significantly increased leaf number, leaf and rosette area, fresh weight and dry weight. Cell size, but typically not cell number, was increased in all tissues resulting in increased vegetative biomass and reproductive organ size, number and seed yield. Ionomic analysis of leaves revealed the VvCEB1opt -overexpressing plants had significantly elevated, K, S and Mo contents relative to control lines. Increased K content likely drives increased osmotic potential within cells leading to greater cellular growth and expansion. To understand the mechanistic basis of VvCEB1opt action, one transgenic line was genotyped using RNA-Seq mRNA expression profiling and revealed a novel transcriptional reprogramming network with significant changes in mRNA abundance for genes with functions in delayed flowering, pathogen-defence responses, iron homeostasis, vesicle-mediated cell wall formation and auxin-mediated signalling and responses. Direct testing of VvCEB1opt -overexpressing plants showed that they had significantly elevated auxin content and a significantly increased number of lateral leaf primordia within meristems relative to controls, confirming that cell expansion and organ number proliferation were likely an auxin-mediated process. VvCEB1opt overexpression in Nicotiana sylvestris also showed larger cells, organ size and biomass demonstrating the potential applicability of this innovative strategy for improving plant biomass and reproductive yield in crops.

The Kalanchoë genome provides insights into convergent evolution and building blocks of crassulacean acid metabolism.

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.

New technologies accelerate the exploration of non-coding RNAs in horticultural plants.

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.

A ζ-carotene desaturase gene, IbZDS, increases ß-carotene and lutein contents and enhances salt tolerance in transgenic sweetpotato.

ζ-Carotene desaturase (ZDS) is one of the key enzymes in carotenoid biosynthesis pathway. However, the ZDS gene has not been applied to carotenoid improvement of plants. Its roles in tolerance to abiotic stresses have not been reported. In this study, the IbZDS gene was isolated from storage roots of sweetpotato (Ipomoea batatas (L.) Lam.) cv. Nongdafu 14. Its overexpression significantly increased ß-carotene and lutein contents and enhanced salt tolerance in transgenic sweetpotato (cv. Kokei No. 14) plants. Significant up-regulation of lycopene ß-cyclase (ß-LCY) and ß-carotene hydroxylase (ß-CHY) genes and significant down-regulation of lycopene ε-cyclase (ε-LCY) and ε-carotene hydroxylase (ε-CHY) genes were found in the transgenic plants. Abscisic acid (ABA) and proline contents and superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) activities were significantly increased, whereas malonaldehyde (MDA) content was significantly decreased in the transgenic plants under salt stress. The salt stress-responsive genes encoding pyrroline-5-carboxylate reductase (P5CR), SOD, CAT, ascorbate peroxidase (APX) and POD were found to be significantly up-regulated in the transgenic plants under salt stress. This study indicates that the IbZDS gene has the potential to be applied for improving ß-carotene and lutein contents and salt tolerance in sweetpotato and other plants.

Advances and perspectives on the use of CRISPR/Cas9 systems in plant genomics research.

Genome editing with site-specific nucleases has become a powerful tool for functional characterization of plant genes and genetic improvement of agricultural crops. Among the various site-specific nuclease-based technologies available for genome editing, the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) systems have shown the greatest potential for rapid and efficient editing of genomes in plant species. This article reviews the current status of application of CRISPR/Cas9 to plant genomics research, with a focus on loss-of-function and gain-of-function analysis of individual genes in the context of perennial plants and the potential application of CRISPR/Cas9 to perturbation of gene expression, and identification and analysis of gene modules as part of an accelerated domestication and synthetic biology effort.

De Novo Transcriptome Sequencing of the Orange-Fleshed Sweet Potato and Analysis of Differentially Expressed Genes Related to Carotenoid Biosynthesis.

Sweet potato, Ipomoea batatas (L.) Lam., is an important food crop worldwide. The orange-fleshed sweet potato is considered to be an important source of beta-carotene. In this study, the transcriptome profiles of an orange-fleshed sweet potato cultivar "Weiduoli" and its mutant "HVB-3" with high carotenoid content were determined by using the high-throughput sequencing technology. A total of 13,767,387 and 9,837,090 high-quality reads were produced from Weiduoli and HVB-3, respectively. These reads were de novo assembled into 58,277 transcripts and 35,909 unigenes with an average length of 596 bp and 533 bp, respectively. In all, 874 differentially expressed genes (DEGs) were obtained between Weiduoli and HVB-3, 401 of which were upregulated and 473 were downregulated in HVB-3 compared to Weiduoli. Of the 697 DEGs annotated, 316 DEGs had GO terms and 62 DEGs were mapped onto 50 pathways. The 22 DEGs and 31 transcription factors involved in carotenoid biosynthesis were identified between Weiduoli and HVB-3. In addition, 1,725 SSR markers were detected. This study provides the genomic resources for discovering the genes involved in carotenoid biosynthesis of sweet potato and other plants.

A Sweetpotato Geranylgeranyl Pyrophosphate Synthase Gene, IbGGPS, Increases Carotenoid Content and Enhances Osmotic Stress Tolerance in Arabidopsis thaliana.

Sweetpotato highly produces carotenoids in storage roots. In this study, a cDNA encoding geranylgeranyl phyrophosphate synthase (GGPS), named IbGGPS, was isolated from sweetpotato storage roots. Green fluorescent protein (GFP) was fused to the C-terminus of IbGGPS to obtain an IbGGPS-GFP fusion protein that was transiently expressed in both epidermal cells of onion and leaves of tobacco. Confocal microscopic analysis determined that the IbGGPS-GFP protein was localized to specific areas of the plasma membrane of onion and chloroplasts in tobacco leaves. The coding region of IbGGPS was cloned into a binary vector under the control of 35S promoter and then transformed into Arabidopsis thaliana to obtain transgenic plants. High performance liquid chromatography (HPLC) analysis showed a significant increase of total carotenoids in transgenic plants. The seeds of transgenic and wild-type plants were germinated on an agar medium supplemented with polyethylene glycol (PEG). Transgenic seedlings grew significantly longer roots than wild-type ones did. Further enzymatic analysis showed an increased activity of superoxide dismutase (SOD) in transgenic seedlings. In addition, the level of malondialdehyde (MDA) was reduced in transgenics. qRT-PCR analysis showed altered expressions of several genes involved in the carotenoid biosynthesis in transgenic plants. These data results indicate that IbGGPS is involved in the biosynthesis of carotenoids in sweetpotato storage roots and likely associated with tolerance to osmotic stress.