To reduce crop losses due to geminivirus infection, we targeted the bean yellow dwarf virus (BeYDV) genome for destruction with the CRISPR–Cas (clustered, regularly interspaced short palindromic repeats–CRISPR-associated proteins) system. Transient assays using BeYDV-based replicons revealed that CRISPR–Cas reagents introduced mutations within the viral genome and reduced virus copy number. Transgenic plants expressing CRISPR–Cas reagents and challenged with BeYDV had reduced virus load and symptoms, thereby demonstrating a novel strategy for engineering resistance to geminiviruses.
Jennifer Mach's insight:
One of a pair of papers in Nature Plants on using CRISPR-Cas for immunity in plants.
Meiosis halves diploid genomes to haploid and is essential for sexual reproduction in eukaryotes. Meiotic recombination ensures physical association of homologs and their subsequent accurate segregation and results in the redistribution of genetic variations among progeny. Most organisms have two classes of cross-overs (COs): interference-sensitive (type I) and -insensitive (type II) COs. DNA synthesis is essential for meiotic recombination, but whether DNA synthesis has a role in differentiating meiotic CO pathways is unknown. Here, we show that Arabidopsis POL2A, the homolog of the yeast DNA polymerase-ε (a leading-strand DNA polymerase), is required for plant fertility and meiosis. Mutations in POL2A cause reduced fertility and meiotic defects, including abnormal chromosome association, improper chromosome segregation, and fragmentation. Observation of prophase I cell distribution suggests that pol2a mutants likely delay progression of meiotic recombination. In addition, the residual COs in pol2a have reduced CO interference, and the double mutant of pol2a with mus81, which affects type II COs, displayed more severe defects than either single mutant, indicating that POL2A functions in the type I pathway. We hypothesize that sufficient leading-strand DNA elongation promotes formation of some type I COs. Given that meiotic recombination and DNA synthesis are conserved in divergent eukaryotes, this study and our previous study suggest a novel role for DNA synthesis in the differentiation of meiotic recombination pathways.
Of the two cultivated species of allopolyploid cotton, Gossypium barbadense produces extra-long fibers for the production of superior textiles. We sequenced its genome (AD)2 and performed a comparative analysis. We identified three bursts of retrotransposons from 20 million years ago (Mya) and a genome-wide uneven pseudogenization peak at 11–20 Mya, which likely contributed to genomic divergences. Among the 2,483 genes preferentially expressed in fiber, a cell elongation regulator,PRE1, is strikingly At biased and fiber specific, echoing the A-genome origin of spinnable fiber. The expansion of the PRE members implies a genetic factor that underlies fiber elongation. Mature cotton fiber consists of nearly pure cellulose. G. barbadense and G. hirsutum contain 29 and 30 cellulose synthase (CesA) genes, respectively; whereas most of these genes (>25) are expressed in fiber, genes for secondary cell wall biosynthesis exhibited a delayed and higher degree of up-regulation inG. barbadense compared with G. hirsutum, conferring an extended elongation stage and highly active secondary wall deposition during extra-long fiber development. The rapid diversification of sesquiterpene synthase genes in the gossypol pathway exemplifies the chemical diversity of lineage-specific secondary metabolites. The G. barbadense genome advances our understanding of allopolyploidy, which will help improve cotton fiber quality.
Physiological aspects of acidity stress in plants (synonymous with H+rhizotoxicity or low-pH stress) have long been a focus of research, in particular with respect to acidic soils where aluminium and H+rhizotoxicities often co-occur. However, toxic H+ and Al3+ elicit different response mechanisms in plants, and it is important to consider their effects separately. The primary aim of this review was to provide the current state of knowledge regarding the genetics of the specific reactions to low-pH stress in growing plants. A comparison of the results gleaned from quantitative trait loci analysis and global transcriptome profiling of plants in response to high proton concentrations revealed a two-stage genetic response: (i) in the short-term, proton pump H+-ATPases present the first barrier in root cells, allocating an excess of H+ into either the apoplast or vacuole; the ensuing defence signaling system involves auxin, salicylic acid, and methyl jasmonate, which subsequently initiate expression of STOP and DREB transcription factors as well as chaperoneROF; (2) the long-term response includes other genes, such as alternative oxidase and type II NAD(P)H dehydrogenase, which act to detoxify dangerous reactive oxygen species in mitochondria, and help plants better manage the stress. A range of transporter genes including those for nitrate (NTR1), malate (ALMT1), and heavy metals are often up-regulated by H+ rhizotoxicity. Expansins, cell-wall-related genes, the γ-aminobutyric acid shunt and biochemical pH-stat genes also reflect changes in cell metabolism and biochemistry in acidic conditions. However, the genetics underlying the acidity stress response of plants is complicated and only fragmentally understood.
Agriculture dominates the planet. Yet it has many environmental costs that are unsustainable, especially as global food demand rises. Here, we evaluate ways in which different parts of the world are succeeding in their attempts to resolve conflict between agriculture and wild nature. We envision that coordinated global action in conserving land most sensitive to agricultural activities and policies that internalise the environmental costs of agriculture are needed to deliver a more sustainable future.
Author Summary The centromeric histone protein, CENH3, plays an important role in chromosome segregation during mitosis and meiosis. Here we show that single amino acid changes in CENH3, while producing no obvious effect on mitosis or meiosis, affect segregation postzygotically upon outcrossing to plants carrying wild-type centromeres. This results in uniparental inheritance among some progeny, and seed death in a larger fraction of progeny. Interestingly, changes competent to induce haploid in Arabidopsis existed in a TILLING population and in unrelated plant species. Our findings have two major consequences. First, uniparental inheritance facilitates the production of haploid plants that can easily be doubled to produce completely homozygous lines in a single generation. Secondly, our findings suggest that natural variation in CENH3 may result in partial reproductive isolation, because chromosomes of the mutant parent from F1 hybrid progeny are culled during embryonic development, while no reproductive defects are observed in self-pollinated plants. We do not know if the same mutations are haploid-inducing in other species, but uniparental chromosome loss, and the seed abortion that accompanies it results in an outcrossing-specific penalty that could potentially be involved in reproductive isolation.
Somaclonal variation arises in plants and animals when differentiated somatic cells are induced into a pluripotent state, but the resulting clones differ from each other and from their parents. In agriculture, somaclonal variation has hindered the micropropagation of elite hybrids and genetically modified crops, but the mechanism responsible remains unknown1. The oil palm fruit ‘mantled’ abnormality is a somaclonal variant arising from tissue culture that drastically reduces yield, and has largely halted efforts to clone elite hybrids for oil production2, 3, 4. Widely regarded as an epigenetic phenomenon5, ‘mantling’ has defied explanation, but here we identify theMANTLED locus using epigenome-wide association studies of the African oil palm Elaeis guineensis. DNA hypomethylation of a LINE retrotransposon related to rice Karma, in the intron of the homeotic gene DEFICIENS, is common to all mantled clones and is associated with alternative splicing and premature termination. Dense methylation near the Karma splice site (termed the Good Karma epiallele) predicts normal fruit set, whereas hypomethylation (the Bad Karma epiallele) predicts homeotic transformation, parthenocarpy and marked loss of yield. Loss of Karma methylation and of small RNA in tissue culture contributes to the origin of mantled, while restoration in spontaneous revertants accounts for non-Mendelian inheritance. The ability to predict and cull mantling at the plantlet stage will facilitate the introduction of higher performing clones and optimize environmentally sensitive land resources.
Maize has a long history of genetic and genomic tool development and is considered one of the most accessible higher plant systems. With a fully sequenced genome, a suite of cytogenetic tools, methods for both forward and reverse genetics, and characterized phenotype markers, maize is amenable to studying questions beyond plant biology. Major discoveries in the areas of transposons, imprinting, and chromosome biology came from work in maize. Moving forward in the post-genomic era, this classic model system will continue to be at the forefront of basic biological study. In this review, we outline the basics of working with maize and describe its rich genetic toolbox.
Distinct modifications fine-tune the activity of jasmonic acid (JA) in regulating plant growth and immunity. Hydroxylated JA (12OH-JA) promotes flower and tuber development but prevents induction of JA signaling, plant defense or both. However, biosynthesis of 12OH-JA has remained elusive. We report here an antibiotic biosynthesis monooxygenase (Abm) that converts endogenous free JA into 12OH-JA in the model rice blast fungus Magnaporthe oryzae. Such fungal 12OH-JA is secreted during host penetration and helps evade the defense response. Loss of Abm in M. oryzae led to accumulation of methyl JA (MeJA), which induces host defense and blocks invasive growth. Exogenously added 12OH-JA markedly attenuated abmΔ-induced immunity in rice. Notably, Abm itself is secreted after invasion and most likely converts plant JA into 12OH-JA to facilitate host colonization. This study sheds light on the chemical arms race during plant-pathogen interaction, reveals Abm as an antifungal target and outlines a synthetic strategy for transformation of a versatile small-molecule phytohormone.
For plant volatile organic compounds (VOCs) to be emitted, they must cross membrane(s), the aqueous cell wall, and sometimes the cuticle, before moving into the gas phase. It is presumed that VOC movement through each barrier occurs via passive diffusion. However, VOCs, which are primarily nonpolar compounds, will preferentially partition into membranes, making diffusion into aqueous compartments slow. Using Fick's first law, we calculated that to achieve observed VOC emission rates by diffusion alone would necessitate toxic VOC levels in membranes. Here, we propose that biological mechanisms, such as those involved in trafficking other hydrophobic compounds, must contribute to VOC emission. Such parallel biological pathways would lower barrier resistances and, thus, steady-state emission rates could be maintained with significantly reduced intramembrane VOC concentrations.
Atmospheric methane is the second most important greenhouse gas after carbon dioxide, and is responsible for about 20% of the global warming effect since pre-industrial times1, 2. Rice paddies are the largest anthropogenic methane source and produce 7–17% of atmospheric methane2, 3. Warm waterlogged soil and exuded nutrients from rice roots provide ideal conditions for methanogenesis in paddies with annual methane emissions of 25–100-million tonnes3, 4. This scenario will be exacerbated by an expansion in rice cultivation needed to meet the escalating demand for food in the coming decades4. There is an urgent need to establish sustainable technologies for increasing rice production while reducing methane fluxes from rice paddies. However, ongoing efforts for methane mitigation in rice paddies are mainly based on farming practices and measures that are difficult to implement5. Despite proposed strategies to increase rice productivity and reduce methane emissions4, 6, no high-starch low-methane-emission rice has been developed. Here we show that the addition of a single transcription factor gene, barleySUSIBA2 (refs 7, 8), conferred a shift of carbon flux to SUSIBA2 rice, favouring the allocation of photosynthates to aboveground biomass over allocation to roots. The altered allocation resulted in an increased biomass and starch content in the seeds and stems, and suppressed methanogenesis, possibly through a reduction in root exudates. Three-year field trials in China demonstrated that the cultivation of SUSIBA2 rice was associated with a significant reduction in methane emissions and a decrease in rhizospheric methanogen levels. SUSIBA2 rice offers a sustainable means of providing increased starch content for food production while reducing greenhouse gas emissions from rice cultivation. Approaches to increase rice productivity and reduce methane emissions as seen in SUSIBA2 rice may be particularly beneficial in a future climate with rising temperatures resulting in increased methane emissions from paddies9, 10.
Deciphering the influence of genetics on primary metabolism in plants will provide insights useful for genetic improvement and enhance our fundamental understanding of plant growth and development. Although maize (Zea mays) is a major crop for food and feed worldwide, the genetic architecture of its primary metabolism is largely unknown. Here, we use high-density linkage mapping to dissect large-scale metabolic traits measured in three different tissues (leaf at seedling stage, leaf at reproductive stage, and kernel at 15 d after pollination [DAP]) of a maize recombinant inbred line population. We identify 297 quantitative trait loci (QTLs) with moderate (86.2% of the mapped QTL, R2 = 2.4 to 15%) to major effects (13.8% of the mapped QTL, R2 >15%) for 79 primary metabolites across three tissues. Pairwise epistatic interactions between these identified loci are detected for more than 25.9% metabolites explaining 6.6% of the phenotypic variance on average (ranging between 1.7 and 16.6%), which implies that epistasis may play an important role for some metabolites. Key candidate genes are highlighted and mapped to carbohydrate metabolism, the tricarboxylic acid cycle, and several important amino acid biosynthetic and catabolic pathways, with two of them being further validated using candidate gene association and expression profiling analysis. Our results reveal a metabolite-metabolite-agronomic trait network that, together with the genetic determinants of maize primary metabolism identified herein, promotes efficient utilization of metabolites in maize improvement.
Crassulacean acid metabolism (CAM) is a specialized mode of photosynthesis that features nocturnal CO2 uptake, facilitates increased water-use efficiency (WUE), and enables CAM plants to inhabit water-limited environments such as semi-arid deserts or seasonally dry forests. Human population growth and global climate change now present challenges for agricultural production systems to increase food, feed, forage, fiber, and fuel production. One approach to meet these challenges is to increase reliance on CAM crops, such as Agave andOpuntia, for biomass production on semi-arid, abandoned, marginal, or degraded agricultural lands. Major research efforts are now underway to assess the productivity of CAM crop species and to harness the WUE of CAM by engineering this pathway into existing food, feed, and bioenergy crops. An improved understanding of CAM has potential for high returns on research investment. To exploit the potential of CAM crops and CAM bioengineering, it will be necessary to elucidate the evolution, genomic features, and regulatory mechanisms of CAM. Field trials and predictive models will be required to assess the productivity of CAM crops, while new synthetic biology approaches need to be developed for CAM engineering. Infrastructure will be needed for CAM model systems, field trials, mutant collections, and data management.
CRISPR–Cas (clustered, regularly interspaced short palindromic repeats–CRISPR-associated proteins) is an adaptive immune system in many archaea and bacteria that cleaves foreign DNA on the basis of sequence complementarity. Here, using the geminivirus, beet severe curly top virus (BSCTV), transient assays performed in Nicotiana benthamiana demonstrate that the sgRNA–Cas9 constructs inhibit virus accumulation and introduce mutations at the target sequences. Further, transgenic Arabidopsis and N. benthamiana plants overexpressing sgRNA–Cas9 are highly resistant to virus infection.
Jennifer Mach's insight:
One of a pair of papers in Nature Plants on using CRISPR-Cas for immunity in plants.
The large brown, round, strongly scented seeds of Ceratocaryum argenteum (Restionaceae) emit many volatiles found to be present in herbivore dung. These seeds attract dung beetles that roll and bury them. As the seeds are hard and offer no reward to the dung beetles, this is a remarkable example of deception in plant seed dispersal.Nature Plants is a scientific journal publishing primary research papers concerned with all aspects of plant biology, technology, ecology and evolution.
Plant resistance genes (R genes) harbor tremendous allelic diversity, constituting a robust immune system effective against microbial pathogens. Nevertheless, few functional R genes have been identified for even the best-studied pathosystems. Does this limited repertoire reflect specificity, with most R genes having been defeated by former pests, or do plants harbor a rich diversity of functional R genes, the composite behavior of which is yet to be characterized? Here, we survey 332 NBS-LRR genes cloned from five resistant Oryza sativa (rice) cultivars for their ability to confer recognition of 12 rice blast isolates when transformed into susceptible cultivars. Our survey reveals that 48.5% of the 132 NBS-LRR loci tested contain functional rice blast R genes, with most R genes deriving from multi-copy clades containing especially diversified loci. Each R gene recognized, on average, 2.42 of the 12 isolates screened. The abundant R genes identified in resistant genomes provide extraordinary redundancy in the ability of host genotypes to recognize particular isolates. If the same is true for other pathogens, many extant NBS-LRR genes retain functionality. Our success at identifying rice blast R genes also validates a highly efficient cloning and screening strategy.
Over 130 years ago, Charles Darwin recognized that sensory functions in the root tip influence directional root growth. Modern plant biology has unravelled that many of the functions that Darwin attributed to the root tip are actually accomplished by a particular organ—the root cap. The root cap surrounds and protects the meristematic stem cells at the growing root tip. Due to this vanguard position, the root cap is predisposed to receive and transmit environmental information to the root proper. In contrast to other plant organs, the root cap shows a rapid turnover of short-lived cells regulated by an intricate balance of cell generation, differentiation, and degeneration. Thanks to these particular features, the root cap is an excellent developmental model system, in which generation, differentiation, and degeneration of cells can be investigated in a conveniently compact spatial and temporal frame. In this review, we give an overview of the current knowledge and concepts of root cap biology, focusing on the model plant Arabidopsis thaliana.
Podophyllotoxin is the natural product precursor of the chemotherapeutic etoposide, yet only part of its biosynthetic pathway is known. We used transcriptome mining in Podophyllum hexandrum(mayapple) to identify biosynthetic genes in the podophyllotoxin pathway. We selected 29 candidate genes to combinatorially express in Nicotiana benthamiana (tobacco) and identified six pathway enzymes, including an oxoglutarate-dependent dioxygenase that closes the core cyclohexane ring of the aryltetralin scaffold. By coexpressing 10 genes in tobacco—these 6 plus 4 previously discovered—we reconstitute the pathway to (–)-4′-desmethylepipodophyllotoxin (the etoposide aglycone), a naturally occurring lignan that is the immediate precursor of etoposide and, unlike podophyllotoxin, a potent topoisomerase inhibitor. Our results enable production of the etoposide aglycone in tobacco and circumvent the need for cultivation of mayapple and semisynthetic epimerization and demethylation of podophyllotoxin.
Thousands of putative biosynthetic genes in Arabidopsis thaliana have no known function, which suggests that there are numerous molecules contributing to plant fitness that have not yet been discovered1, 2. Prime among these uncharacterized genes are cytochromes P450 upregulated in response to pathogens3, 4. Here we start with a single pathogen-induced P450 (ref. 5), CYP82C2, and use a combination of untargeted metabolomics and coexpression analysis to uncover the complete biosynthetic pathway to 4-hydroxyindole-3-carbonyl nitrile (4-OH-ICN), a previously unknown Arabidopsis metabolite. This metabolite harbours cyanogenic functionality that is unprecedented in plants and exceedingly rare in nature6, 7; furthermore, the aryl cyanohydrin intermediate in the 4-OH-ICN pathway reveals a latent capacity for cyanogenic glucoside biosynthesis8, 9 in Arabidopsis. By expressing 4-OH-ICN biosynthetic enzymes in Saccharomyces cerevisiae and Nicotiana benthamiana, we reconstitute the complete pathway in vitro and in vivoand validate the functions of its enzymes. Arabidopsis 4-OH-ICN pathway mutants show increased susceptibility to the bacterial pathogen Pseudomonas syringae, consistent with a role in inducible pathogen defence. Arabidopsis has been the pre-eminent model system10, 11 for studying the role of small molecules in plant innate immunity12; our results uncover a new branch of indole metabolism distinct from the canonical camalexin pathway, and support a role for this pathway in the Arabidopsis defence response13. These results establish a more complete framework for understanding how the model plant Arabidopsis uses small molecules in pathogen defence.
The plant hormone jasmonate plays crucial roles in regulating plant responses to herbivorous insects and microbial pathogens and is an important regulator of plant growth and development1,2, 3, 4, 5, 6, 7. Key mediators of jasmonate signalling include MYC transcription factors, which are repressed by jasmonate ZIM-domain (JAZ) transcriptional repressors in the resting state. In the presence of active jasmonate, JAZ proteins function as jasmonate co-receptors by forming a hormone-dependent complex with COI1, the F-box subunit of an SCF-type ubiquitin E3 ligase8, 9,10, 11. The hormone-dependent formation of the COI1–JAZ co-receptor complex leads to ubiquitination and proteasome-dependent degradation of JAZ repressors and release of MYC proteins from transcriptional repression3, 10, 12. The mechanism by which JAZ proteins repress MYC transcription factors and how JAZ proteins switch between the repressor function in the absence of hormone and the co-receptor function in the presence of hormone remain enigmatic. Here we show that Arabidopsis MYC3 undergoes pronounced conformational changes when bound to the conserved Jas motif of the JAZ9 repressor. The Jas motif, previously shown to bind to hormone as a partly unwound helix, forms a complete α-helix that displaces the amino (N)-terminal helix of MYC3 and becomes an integral part of the MYC N-terminal fold. In this position, the Jas helix competitively inhibits MYC3 interaction with the MED25 subunit of the transcriptional Mediator complex. Our structural and functional studies elucidate a dynamic molecular switch mechanism that governs the repression and activation of a major plant hormone pathway.
In plants, genomic DNA methylation which contributes to development and stress responses can be actively removed by DEMETER-like DNA demethylases (DMLs). Indeed, in Arabidopsis DMLs are important for maternal imprinting and endosperm demethylation, but only a few studies demonstrate the developmental roles of active DNA demethylation conclusively in this plant. Here, we show a direct cause and effect relationship between active DNA demethylation mainly mediated by the tomato DML, SlDML2, and fruit ripening— an important developmental process unique to plants. RNAi SlDML2 knockdown results in ripening inhibition via hypermethylation and repression of the expression of genes encoding ripening transcription factors and rate-limiting enzymes of key biochemical processes such as carotenoid synthesis. Our data demonstrate that active DNA demethylation is central to the control of ripening in tomato.
Maize, the highest-yielding cereal crop worldwide, is particularly susceptible to drought during its 2- to 3-week flowering period. Many genetic engineering strategies for drought tolerance impinge on plant development, reduce maximum yield potential or do not translate from laboratory conditions to the field. We overexpressed a gene encoding a rice trehalose-6-phosphate phosphatase (TPP) in developing maize ears using a floral promoter. This reduced the concentration of trehalose-6-phosphate (T6P), a sugar signal that regulates growth and development, and increased the concentration of sucrose in ear spikelets. Overexpression of TPP increased both kernel set and harvest index. Field data at several sites and over multiple seasons showed that the engineered trait improved yields from 9% to 49% under non-drought or mild drought conditions, and from 31% to 123% under more severe drought conditions, relative to yields from nontransgenic controls.
teosinte glume architecture1 (tga1), a member of the SBP-box gene family of transcriptional regulators, has been identified as the gene conferring naked kernels in maize vs. encased kernels in its wild progenitor, teosinte. However, the identity of the causative polymorphism within tga1 that produces these different phenotypes has remained unknown. Using nucleotide diversity data, we show that there is a single fixed nucleotide difference between maize and teosinte in tga1, and this difference confers a Lys (teosinte allele) to Asn (maize allele) substitution. This substitution transforms TGA1 into a transcriptional repressor. While both alleles of TGA1 can bind a GTAC motif, maize-TGA1 forms more stable dimers than teosinte-TGA1. Since it is the only fixed difference between maize and teosinte, this alteration in protein function likely underlies the differences in maize and teosinte glume architecture. We previously reported a difference in TGA1 protein abundance between maize and teosinte based on relative signal intensity of a Western blot. Here, we show that this signal difference is not due to tga1 but to a second gene, neighbor of tga1 (not1). Not1encodes a protein that has 92% amino acid similarity to TGA1 and that is recognized by the TGA1 antibody. Genetic mapping and phenotypic data show that tga1, without a contribution from not1, controls the difference in covered vs. naked kernels. No trait differences could be associated with the maize vs. teosinte alleles of not1. Our results document how morphological evolution can be driven by a simple nucleotide change that alters protein function.
Mazzoleni et al. (2015a,b) in two recent papers provided novel and rigorous evidence for a unique detrimental effect of self-DNA (i.e. DNA originating from conspecifics) on organismal growth. The authors investigated the effect as a means of explaining plant–soil feedbacks via plant litter (Mazzoleni et al., 2015a) and subsequently convincingly generalized their observations to a range of additional organisms including protozoa, algae, fungi and animals. The authors explain the growth suppression on the basis of inhibitory effects of self-DNA. They argue that this inhibition mechanism, through facilitating coexistence, represents a mechanism of maintaining diversity. The ecological, physiological and molecular significance of the observations of Mazzoleni et al. (2015a,b) is thought-provoking. A priority now is to start a discourse on the interpretation of the results of these studies, because this will help design focused experiments to further investigate the role of self-DNA on growth.
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