JGI Plant Gene Atlas: an updateable transcriptome resource to improve functional gene descriptions across the plant kingdom.

Gene functional descriptions offer a crucial line of evidence for candidate genes underlying trait variation. Conversely, plant responses to environmental cues represent important resources to decipher gene function and subsequently provide molecular targets for plant improvement through gene editing. However, biological roles of large proportions of genes across the plant phylogeny are poorly annotated. Here we describe the Joint Genome Institute (JGI) Plant Gene Atlas, an updateable data resource consisting of transcript abundance assays spanning 18 diverse species. To integrate across these diverse genotypes, we analyzed expression profiles, built gene clusters that exhibited tissue/condition specific expression, and tested for transcriptional response to environmental queues. We discovered extensive phylogenetically constrained and condition-specific expression profiles for genes without any previously documented functional annotation. Such conserved expression patterns and tightly co-expressed gene clusters let us assign expression derived additional biological information to 64 495 genes with otherwise unknown functions. The ever-expanding Gene Atlas resource is available at JGI Plant Gene Atlas (https://plantgeneatlas.jgi.doe.gov) and Phytozome (https://phytozome.jgi.doe.gov/), providing bulk access to data and user-specified queries of gene sets. Combined, these web interfaces let users access differentially expressed genes, track orthologs across the Gene Atlas plants, graphically represent co-expressed genes, and visualize gene ontology and pathway enrichments.

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.

Access to Bromo-γ-butenolides via Zwitterion-Catalyzed Rearrangement of Cyclopropene Carboxylic Acids.

γ-Butenolides are useful structural motifs in many pharmaceutically relevant compounds. In particular, halogenated γ-butenolides are attractive building blocks because the halogen handles can readily be manipulated to give various functional molecules. In this study, a catalytic synthesis of halogenated γ-butenolides from cyclopropene carboxylic acids was developed using zwitterionic catalysts and N-haloamides as the halogen sources. The catalytic protocol could also be applied to the synthesis of halogenated pyrrolones by using cyclopropene amides as the starting materials.

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.

Cultivated and Wild Grapevines in Tennessee Possess Overlapping but Distinct Virus Populations.

Viruses and viroids prevalent in a population of 42 wild grapevines (i.e., free-living, uncultivated grapevines; Vitis spp.) were compared with those in a population of 85 cultivated grapevines collected in Tennessee, United States by RNA sequencing analysis of pools of ribosomal RNA-depleted total RNA. The sequences of 10 viruses (grapevine fleck virus, grapevine leafroll-associated virus 2, grapevine rupestris stem pitting-associated virus, grapevine Syrah virus 1, grapevine vein-clearing virus, grapevine virus B, grapevine virus E, tobacco ringspot virus, tomato ringspot virus, and a novel nano-like virus) and two viroids (hop stunt viroid and grapevine yellow speckle viroid 1) were detected in both grapevine populations. Sequences of four viruses (grapevine associated tymo-like virus, grapevine leafroll-associated virus 3, grapevine red blotch virus, and grapevine virus H) were identified only from cultivated grapevines. High, moderate, and low numbers of sequence reads were identified only from wild grapevines for a novel caulimovirus, an enamovirus, and alfalfa mosaic virus, respectively. The presence of most virus sequences and both viroids was verified independently in the original samples by reverse-transcription PCR followed by Sanger sequencing. Comparison of viral sequences shared by both populations showed that cultivated and wild grapevines harbored distinct sequence variants, which suggests that there was limited virus movement between the two populations. Collectively, this study represents the first unbiased survey of viruses and viroids in both cultivated and wild grapevines within a defined geographic region.

Zwitterion-Catalyzed Intermolecular Bromoesterifications.

Intermolecular haloesterification is an important class of transformations. The resulting products are valuable building blocks. However, it is often necessary to use super-stoichiometric amount of acid in order to compensate the low reactivity. Herein, we report a zwitterion-catalyzed intermolecular bromoesterification using acid and olefin in an equimolar ratio. Mechanistic study revealed that the charge pair in the zwitterion works synergistically in activating both NBS and carboxylic acid.

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.

AKR2A participates in the regulation of cotton fibre development by modulating biosynthesis of very-long-chain fatty acids.

The biosynthesis of very-long-chain fatty acids (VLCFAs) and their transport are required for fibre development. However, whether other regulatory factors are involved in this process is unknown. We report here that overexpression of an Arabidopsis gene ankyrin repeat-containing protein 2A (AKR2A) in cotton promotes fibre elongation. RNA-Seq analysis was employed to elucidate the mechanisms of AKR2A in regulating cotton fibre development. The VLCFA content and the ratio of VLCFAs to short-chain fatty acids increased in AKR2A transgenic lines. In addition, AKR2A promotes fibre elongation by regulating ethylene and synergizing with the accumulation of auxin and hydrogen peroxide. Analysis of RNA-Seq data indicates that AKR2A up-regulates transcript levels of genes involved in VLCFAs' biosynthesis, ethylene biosynthesis, auxin and hydrogen peroxide signalling, cell wall and cytoskeletal organization. Furthermore, AKR2A interacted with KCS1 in Arabidopsis both in vitro and in vivo. Moreover, the VLCFA content and the ratio of VLCFAs to short-chain fatty acids increased significantly in seeds of AKR2A-overexpressing lines and AKR2A/KCS1 co-overexpressing lines, while AKR2A mutants are the opposite trend. Our results uncover a novel cotton fibre growth mechanism by which the critical regulator AKR2A promotes fibre development via activating hormone signalling cascade by mediating VLCFA biosynthesis. This study provides a potential candidate gene for improving fibre yield and quality through genetic engineering.

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.

Identification of Populus Small RNAs Responsive to Mutualistic Interactions With Mycorrhizal Fungi, Laccaria bicolor and Rhizophagus irregularis.

Ecto- and endo-mycorrhizal colonization of Populus roots have a positive impact on the overall tree health and growth. 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. 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. 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 differentially-expressed 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. 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.

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 yield difference between wild-type cotton and transgenic cotton that expresses IPT depends on when water-deficit stress is applied.

Drought is the No. 1 factor that limits agricultural production in the world, thus, making crops more drought tolerant is a major goal in agriculture. Many genes with functions in abiotic stress tolerance were identified, and overexpression of these genes confers increased drought tolerance in transgenic plants. The isopentenyltransferase gene (IPT) that encodes a rate limiting enzyme in cytokinin biosynthesis is one of them. Interestingly, when IPT-transgenic cotton was field-tested at two different sites, Texas and Arizona, different results were obtained. To explain this phenomenon, reduced irrigation experiments with different timing in applying water deficit stress were conducted. It was found that the timing of water deficit stress is critical for IPT-transgenic cotton to display its yield advantage over control plants (i.e. wild-type and segregated non-transgenic plants). If water deficit stress occurs before flowering (vegetative phase), IPT-transgenic cotton would outperform control plants; however, if water deficit stress occurs at or after flowering (reproductive phase), there would not be a yield difference between IPT-transgenic and control cotton plants. This result suggests that an early induction of IPT expression (before first flowering) is needed in order to realize the benefits of IPT-expression in transgenic plants that face water-deficit stress later in development.

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.

Visible-Light-Induced Carbo-2-pyridylation of Electron-Deficient Alkenes with Pyridinium Salts.

A simple and practical visible-light-induced carbo-2-pyridylation of electron-deficient alkenes with readily available N-benzoylmethylpyridinium bromides is reported. More than 40 examples are presented and proceed in greater than 80 % yield (on average) with excellent regio- and diastereoselectivities.

Populus trichocarpa encodes small, effector-like secreted proteins that are highly induced during mutualistic symbiosis.

During symbiosis, organisms use a range of metabolic and protein-based signals to communicate. Of these protein signals, one class is defined as 'effectors', i.e., small secreted proteins (SSPs) that cause phenotypical and physiological changes in another organism. To date, protein-based effectors have been described in aphids, nematodes, fungi and bacteria. Using RNA sequencing of Populus trichocarpa roots in mutualistic symbiosis with the ectomycorrhizal fungus Laccaria bicolor, we sought to determine if host plants also contain genes encoding effector-like proteins. We identified 417 plant-encoded putative SSPs that were significantly regulated during this interaction, including 161 SSPs specific to P. trichocarpa and 15 SSPs exhibiting expansion in Populus and closely related lineages. We demonstrate that a subset of these SSPs can enter L. bicolor hyphae, localize to the nucleus and affect hyphal growth and morphology. We conclude that plants encode proteins that appear to function as effector proteins that may regulate symbiotic associations.

Overexpression of the PP2A-C5 gene confers increased salt tolerance in Arabidopsis thaliana.

Protein phosphatase 2A (PP2A) was shown to play important roles in biotic and abiotic stress signaling pathways in plants. PP2A is made of 3 subunits: a scaffolding subunit A, a regulatory subunit B, and a catalytic subunit C. It is believed that the B subunit recognizes specific substrates and the C subunit directly acts on the selected substrates, whereas the A subunit brings a B subunit and a C subunit together to form a specific PP2A holoenzyme. Because there are multiple isoforms for each PP2A subunit, there could be hundreds of novel PP2A holoenzymes in plants. For an example, there are 3 A subunits, 17 B subunits, and 5 C subunits in Arabidopsis, which could form 255 different PP2A holoenzymes. Understanding the roles of these PP2A holoenzymes in various signaling pathways is a challenging task. In a recent study, 1 we discovered that PP2A-C5, the catalytic subunit 5 of PP2A, plays an important role in salt tolerance in Arabidopsis. We found that a knockout mutant of PP2A-C5 (i.e. pp2a-c5-1) was very sensitive to salt treatments, whereas PP2A-C5-overexpressing plants were more tolerant to salt stresses. Genetic analyses between pp2a-c5-1 and Salt-Overly-Sensitive (SOS) mutants indicated that PP2A-C5 does not function in the same pathway as SOS genes. Using yeast 2-hybrid analysis, we found that PP2A-C5 interacts with several vacuolar membrane bound chloride channel proteins. We hypothesize that these vacuolar chloride channel proteins might be PP2A-C5's substrates in vivo, and the action of PP2A-C5 on these channel proteins could increase or activate their activities, thereby result in accumulation of the chloride and sodium contents in vacuoles, leading to increased salt tolerance in plants.

Overexpression of PP2A-C5 that encodes the catalytic subunit 5 of protein phosphatase 2A in Arabidopsis confers better root and shoot development under salt conditions.

Protein phosphatase 2A (PP2A) is an enzyme consisting of three subunits: a scaffolding A subunit, a regulatory B subunit and a catalytic C subunit. PP2As were shown to play diverse roles in eukaryotes. In this study, the function of the Arabidopsis PP2A-C5 gene that encodes the catalytic subunit 5 of PP2A was studied using both loss-of-function and gain-of-function analyses. Loss-of-function mutant pp2a-c5-1 displayed more impaired growth during root and shoot development, whereas overexpression of PP2A-C5 conferred better root and shoot growth under different salt treatments, indicating that PP2A-C5 plays an important role in plant growth under salt conditions. Double knockout mutants of pp2a-c5-1 and salt overly sensitive (sos) mutants sos1-1, sos2-2 or sos3-1 showed additive sensitivity to NaCl, indicating that PP2A-C5 functions in a pathway different from the SOS signalling pathway. Using yeast two-hybrid analysis, four vacuolar membrane chloride channel (CLC) proteins, AtCLCa, AtCLCb, AtCLCc and AtCLCg, were found to interact with PP2A-C5. Moreover, overexpression of AtCLCc leads to increased salt tolerance and Cl- accumulation in transgenic Arabidopsis plants. These data indicate that PP2A-C5-mediated better growth under salt conditions might involve up-regulation of CLC activities on vacuolar membranes and that PP2A-C5 could be used for improving salt tolerance in crops.