This new volume of Methods in Enzymology continues the legacy of this premier serial with quality chapters authored by leaders in the field. Methods to assess mitochondrial function is of great interest to neuroscientists studying chronic forms of neurodegeneration, including Parkinson's, Alzheimer's, ALS, Huntington's and other triplet repeat diseases, but also to those working on acute conditions such as stroke and traumatic brain injury. This volume covers research methods on how to assess the life cycle of mitochondria including trafficking, fusion, fission, and degradation. Multiple perspectives on the complex and difficult problem of measurement of mitochondrial reactive oxygen species production with fluorescent indicators and techniques ranging in scope from measurements on isolated mitochondria to non-invasive imaging of metabolic function.Continues the legacy of this premier serial with quality chapters authored by leaders in the fieldCovers research methods in biomineralization scienceProvides invaluable details on state-of-the-art methods to assess a broad array of mitochondrial functions
Recent advances in the targeted modification of complex eukaryotic genomes have unlocked a new era of genome engineering. From the pioneering work using zinc-finger nucleases (ZFNs), to the advent of the versatile and specific TALEN systems, and most recently the highly accessible CRISPR/Cas9 systems, we now possess an unprecedented ability to analyze developmental processes using sophisticated designer genetic tools. Excitingly, these robust and simple genomic engineering tools also promise to revolutionize developmental studies using less well established experimental organisms.
Modern developmental biology was born out of the fruitful marriage between traditional embryology and genetics. Genetic tools, together with advanced microscopy techniques, serve as the most fundamental means for developmental biologists to elucidate the logistics and the molecular control of growth, differentiation and morphogenesis. For this reason, model organisms with sophisticated and comprehensive genetic tools have been highly favored for developmental studies. Advances made in developmental biology using these genetically amenable models have been well recognized. The Nobel prize in Physiology or Medicine was awarded in 1995 to Edward B. Lewis, Christiane Nüsslein-Volhard and Eric F. Wieschaus for their discoveries on the ‘Genetic control of early structural development’ usingDrosophila melanogaster, and again in 2002 to John Sulston, Robert Horvitz and Sydney Brenner for their discoveries of ‘Genetic regulation of development and programmed cell death’ using the nematode worm Caenorhabditis elegans. These fly and worm systems remain powerful and popular models for invertebrate development studies, while zebrafish (Danio rerio), the dual frog species Xenopus laevis and Xenopus tropicalis, rat (Rattus norvegicus), and particularly mouse (Mus musculus) represent the most commonly used vertebrate model systems. To date, random or semi-random mutagenesis (‘forward genetic’) approaches have been extraordinarily successful at advancing the use of these model organisms in developmental studies. With the advent of reference genomic data, however, sequence-specific genomic engineering tools (‘reverse genetics’) enable targeted manipulation of the genome and thus allow previously untestable hypotheses of gene function to be addressed.
Communication is an integral part of the research you perform as a scientist. Your written papers serve as a gauge of your scientific productivity and provide a long-lasting body of knowledge from which other scientists can build their research. The oral presentations you deliver make your latest research known to the community, helping your peers stay up to date. Discussions enable you to exchange ideas and points of view. Letters, memos, and résumés help you build and maintain relationships with colleagues, suppliers, employers, and so on.
The discovery of high-temperature superconductors, the determination of DNA’s double-helix structure, the first observations that the expansion of the Universe is accelerating — all of these breakthroughs won Nobel prizes and international acclaim. Yet none of the papers that announced them comes anywhere close to ranking among the 100 most highly cited papers of all time.
Citations, in which one paper refers to earlier works, are the standard means by which authors acknowledge the source of their methods, ideas and findings, and are often used as a rough measure of a paper’s importance. Fifty years ago, Eugene Garfield published the Science Citation Index (SCI), the first systematic effort to track citations in the scientific literature. To mark the anniversary, Nature asked Thomson Reuters, which now owns the SCI, to list the 100 most highly cited papers of all time. (See the full list at Web of Science Top 100.xls or the interactive graphic, below.) The search covered all of Thomson Reuter’s Web of Science, an online version of the SCI that also includes databases covering the social sciences, arts and humanities, conference proceedings and some books. It lists papers published from 1900 to the present day.
The exercise revealed some surprises, not least that it takes a staggering 12,119 citations to rank in the top 100 — and that many of the world’s most famous papers do not make the cut. A few that do, such as the first observation1 of carbon nanotubes (number 36) are indeed classic discoveries. But the vast majority describe experimental methods or software that have become essential in their fields.
The most cited work in history, for example, is a 1951 paper2 describing an assay to determine the amount of protein in a solution. It has now gathered more than 305,000 citations — a recognition that always puzzled its lead author, the late US biochemist Oliver Lowry.
Plant microbiomes are critical to host adaptation and impact plant productivity and health. Root-associated microbiomes vary by soil and host genotype, but the contribution of these factors to community structure and metabolic potential has not been fully addressed. Here we characterize root microbial communities of two disparate agricultural crops grown in the same natural soil in a controlled and replicated experimental system. Metagenomic (genetic potential) analysis identifies a core set of functional genes associated with root colonization in both plant hosts, and metatranscriptomic (functional expression) analysis revealed that most genes enriched in the root zones are expressed. Root colonization requires multiple functional capabilities, and these capabilities are enriched at the community level. Differences between the root-associated microbial communities from different plants are observed at the genus or species level, and are related to root-zone environmental factors.
In crop improvement, the isolation, cloning and transfer of disease resistance genes (R-genes) is an ultimate goal usually starting from tentative R-gene analogs (RGAs) that are identified on the basis of their structure. For bread wheat, recent advances in genome sequencing are supporting the efforts of wheat geneticists worldwide. Among wheat R-genes, nucleotide-binding site (NBS)-encoding ones represent a major class. In this study, we have used a polymerase chain reaction-based approach to amplify and clone NBS-type RGAs from a bread wheat cultivar, ‘Salambo 80.’ Four novel complete ORF sequences showing similarities to previously reported R-genes/RGAs were used for in silico analyses. In a first step, where analyses were focused on the NBS domain, these sequences were phylogenetically assigned to two distinct groups: a first group close to leaf rust Lr21 resistance proteins; and a second one similar to cyst nematode resistance proteins. In a second step, sequences were used as initial seeds to walk up and downstream the NBS domain. This procedure enabled identifying 8 loci ranging in size between 2,115 and 7,653 bp. Ab initio gene prediction identified 8 gene models, among which two had complete ORFs. While GenBank survey confirmed the belonging of sequences to two groups, subsequent characterization using IWGSC genomic and proteomic data showed that the 8 gene models, reported in this study, were unique and their loci matched scaffolds on chromosome arms 1AS, 1BS, 4BS and 1DS. The gene model located on 1DS is a pseudo-Lr21 that was shown to have an NBS-LRR domain structure, while the potential association of the RGAs, here reported, is discussed. This study has produced novel R-gene-like loci and models in the wheat genome and provides the first steps toward further elucidation of their role in wheat disease resistance.
Genome sequencing projects were long confined to biomedical model organisms and required the concerted effort of large consortia. Rapid progress in high-throughput sequencing technology and the simultaneous development of bioinformatic tools have democratized the field. It is now within reach for individual research groups in the eco-evolutionary and conservation community to generate de novo draft genome sequences for any organism of choice. Because of the cost and considerable effort involved in such an endeavour, the important first step is to thoroughly consider whether a genome sequence is necessary for addressing the biological question at hand. Once this decision is taken, a genome project requires careful planning with respect to the organism involved and the intended quality of the genome draft. Here, we briefly review the state of the art within this field and provide a step-by-step introduction to the workflow involved in genome sequencing, assembly and annotation with particular reference to large and complex genomes. This tutorial is targeted at scientists with a background in conservation genetics, but more generally, provides useful practical guidance for researchers engaging in whole-genome sequencing projects.
Scott Jackson, Jeremy Schmutz, Phillip McClean and colleagues report the genome sequence of the common bean (Phaseolus vulgaris) and resequenced wild individuals and landraces from Mesoamerican and Andean gene pools, showing that common bean underwent two independent domestications.
Two classes of genes are used for breeding rust resistant wheat. The first class, called R (for resistance) genes, are pathogen race-specific in their action, effective at all plant growth stages and probably mostly encode immune receptors of the nucleotide binding leucine rich repeat (NB-LRR) class. The second class called Adult Plant Resistance genes (APR) because resistance is usually functional only in adult plants, and, in contrast to most R genes, the levels of resistance conferred by single APR genes are only partial and allow considerable disease development. Some but not all APR genes provide resistance to all isolates of a rust pathogen species and a subclass of these provides resistance to several fungal pathogen species. Initial indications are that APR genes encode a more heterogeneous range of proteins than R proteins. Two APR genes, Lr34 and Yr36, have been cloned from wheat and their products are an ABC transporter and a protein kinase, respectively. Lr34 and Sr2 have provided long lasting and widely used (durable) partial resistance and are mainly used in conjunction with other R and APR genes to obtain adequate rust resistance. We caution that some APR genes indeed include race-specific, weak R genes which may be of the NB-LRR class. A research priority to better inform rust resistance breeding is to characterize further APR genes in wheat and to understand how they function and how they interact when multiple APR and R genes are stacked in a single genotype by conventional and GM breeding. An important message is do not be complacent about the general durability of all APR genes.
Nine billion people are expected to inhabit Planet Earth by 2050. Without agricultural research, there is little hope of sustaining this population surge, given that arable land and water supplies are fixed commodities. Yet for decades the agricultural sector has suffered from neglect. If we want to combat new strains of pests that destroy crops, find new crop varieties enriched in nutritional value, improve yields, develop resistance to disease and drought, and provide environmentally sensitive cultivation practices, then agricultural research must be a priority. Why isn't it?
In photosynthetic organisms, D-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the major enzyme assimilating atmospheric CO2 into the biosphere1. Owing to the wasteful oxygenase activity and slow turnover of Rubisco, the enzyme is among the most important targets for improving the photosynthetic efficiency of vascular plants2, 3. It has been anticipated that introducing the CO2-concentrating mechanism (CCM) from cyanobacteria into plants could enhance crop yield4, 5, 6. However, the complex nature of Rubisco’s assembly has made manipulation of the enzyme extremely challenging, and attempts to replace it in plants with the enzymes from cyanobacteria and red algae have not been successful7, 8. Here we report two transplastomic tobacco lines with functional Rubisco from the cyanobacterium Synechococcus elongatus PCC7942 (Se7942). We knocked out the native tobacco gene encoding the large subunit of Rubisco by inserting the large and small subunit genes of the Se7942 enzyme, in combination with either the corresponding Se7942 assembly chaperone, RbcX, or an internal carboxysomal protein, CcmM35, which incorporates three small subunit-like domains9, 10. Se7942 Rubisco and CcmM35 formed macromolecular complexes within the chloroplast stroma, mirroring an early step in the biogenesis of cyanobacterial β-carboxysomes11, 12. Both transformed lines were photosynthetically competent, supporting autotrophic growth, and their respective forms of Rubisco had higher rates of CO2 fixation per unit of enzyme than the tobacco control. These transplastomic tobacco lines represent an important step towards improved photosynthesis in plants and will be valuable hosts for future addition of the remaining components of the cyanobacterial CCM, such as inorganic carbon transporters and the β-carboxysome shell proteins
Bacteria are able to sense their population's density through a cell–cell communication system, termed ‘quorum sensing’ (QS). This system regulates gene expression in response to cell density through the constant production and detection of signalling molecules. These molecules commonly act as auto-inducers through the up-regulation of their own synthesis. Many pathogenic bacteria, including those of plants, rely on this communication system for infection of their hosts. The finding that the countering of QS-disrupting mechanisms exists in many prokaryotic and eukaryotic organisms offers a promising novel method to fight disease. During the last decade, several approaches have been proposed to disrupt QS pathways of phytopathogens, and hence to reduce their virulence. Such studies have had varied success in vivo, but most lend promising support to the idea that QS manipulation could be a potentially effective method to reduce bacterial-mediated plant disease. This review discusses the various QS-disrupting mechanisms found in both bacteria and plants, as well as the different approaches applied artificially to interfere with QS pathways and thus protect plant health.
Knockout of all six alleles of a gene in the large wheat genome confers resistance to powdery mildew --- Genetic engineering to improve crops is entering a new era as conventional transgenesis technology, which involves random insertion of genes into the genome, is superseded by newer approaches that enable precise genetic alterations. A particular technological challenge in carrying out targeted genome modification in crops is that many plant genomes are polyploid, including such important species as wheat, potato and canola1. In this issue, Wang et al.2 report engineering of the hexaploid wheat genome using sequence-specific nucleases (SSNs)—the first demonstration in a polyploid crop of SSN-mediated genetic alterations that are stably transmitted to the next generation. By knocking out all six alleles encoding the MILDEW-RESISTANCE LOCUS (MLO) protein, the authors generated a mutant line that shows strong resistance to powdery mildew, a devastating fungal disease. This is a remarkable feat, given the ploidy and enormous size (17.1 Gb) of the wheat genome, and showcases the power of SSNs for engineering complex plant genomes and for creating crops with valuable traits.
Plant interactions with other organisms: molecules, ecology and evolution
Different shades of JAZ during plant growth and defense
Nutrient supply differentially alters the dynamics of co-infecting phytoviruses
From shade avoidance responses to plant performance at vegetation level: using virtual plant modelling as a tool F. J. Bongers, J. B. Evers, N. P. R. Anten & R. Pierik
Magical mystery tour: MLO proteins in plant immunity and beyond J. Acevedo-Garcia, S. Kusch & R. Panstruga
The squeeze cell hypothesis for the activation of jasmonate synthesis in response to wounding
E. E. Farmer, D. Gasperini & I. F. Acosta
Lipochitooligosaccharide recognition: an ancient story Y. Liang, K. Tóth, Y. Cao, K. Tanaka, C. Espinoza & G. Stacey
Herbivore-induced plant volatiles: targets, perception and unanswered questions M. Heil
There’s no place like home? An exploration of the mechanisms behind plant litter–decomposer affinity in terrestrial ecosystems A. T. Austin, L. Vivanco, A. González-Arzac & L. I. Pérez
Insect herbivore-associated organisms affect plant responses to herbivory F. Zhu, E. H. Poelman & M. Dicke
When mutualism goes bad: density- dependent impacts of introduced bees on plant reproduction M. A. Aizen, C. L. Morales, D. P. Vázquez, L. A. Garibaldi, A. Sáez & L. D. Harder
Insect and pathogen attack and resistance in maize and its wild ancestors, the teosintes E. S. de Lange, D. Balmer, B. Mauch-Mani & T. C. J. Turlings
Linking phytochrome to plant immunity: low red : far-red ratios increase Arabidopsis susceptibility to Botrytis cinerea by reducing the biosynthesis of indolic glucosinolates and camalexin M. D. Cargnel, P. V. Demkura & C. L. Ballaré
To grow or defend? Low red : far-red ratios reduce jasmonate sensitivity in Arabidopsis seedlings by promoting DELLA degradation and increasing JAZ10 stability M. Leone, M. M. Keller, I. Cerrudo & C. L. Ballaré
β-Glucosidase BGLU42 is a MYB72-dependent key regulator of rhizobacteria-induced systemic resistance and modulates iron deficiency responses in Arabidopsis roots C. Zamioudis, J. Hanson & C. M. J. Pieterse
Deciphering the language of plant communication: volatile chemotypes of sagebrush R. Karban, W. C. Wetzel, K. Shiojiri, S. Ishizaki, S. R. Ramirez & J. D. Blande
The context dependence of beneficiary feedback effects on benefactors in plant facilitation C. Schöb, R. M. Callaway, F. Anthelme, R. W. Brooker, L. A. Cavieres, Z. Kikvidze, C. J. Lortie, R. Michalet, F. I. Pugnaire, S. Xiao, B. H. Cranston, M-C. García, N. R. Hupp, L. D. Llambí, E. Lingua, A. M. Reid, L. Zhao & B. J. Butterfield
Herbivore-mediated material fluxes in a northern deciduous forest under elevated carbon dioxide and ozone concentrations T. D. Meehan, J. J. Couture, A. E. Bennett & R. L. Lindroth
Are plant–soil feedback responses explained by plant traits? C. Baxendale, K. H. Orwin, F. Poly, T. Pommier & R. D. Bardgett
Environmental nutrient supply alters prevalence and weakens competitive interactions among coinfecting viruses C. Lacroix, E. W. Seabloom & E. T. Borer
Scientists from the Wyss Institute, the Broad Institute, and the University of California, Berkeley, have helped develop a versatile and powerful new genome editing tool by altering a natural method of bacterial self-defense. Bacteria use a system called CRISPR to store DNA from invading viruses so they can recognize and cleave that foreign DNA later when the invader returns. CRISPR-associated (Cas) enzymes do the cutting, and one in particular, an enzyme from the bacterium Streptococcus pyogenes known as Cas9, can cut DNA at exactly the location dictated by an RNA — a snippet of RNA that can be programmed to seek out precise DNA sequences in the genome to edit. Wyss Institute scientists have shown for the first time that the Cas9 system works in cells of higher organisms, including humans. The CRISPR/Cas9 technology enables highly efficient and specific genome editing, opening the path to numerous therapeutic applications such as correcting genetic disorders and battling invading pathogens.
Millions of tons. That’s how much plastic should be floating in the world’s oceans, given our ubiquitous use of the stuff. But a new study finds that 99% of this plastic is missing. One disturbing possibility: Fish are eating it.
If that’s the case, “there is potential for this plastic to enter the global ocean food web,” says Carlos Duarte, an oceanographer at the University of Western Australia, Crawley. “And we are part of this food web.”
Humans produce almost 300 million tons of plastic each year. Most of this ends up in landfills or waste pits, but a 1970s National Academy of Sciences study estimated that 0.1% of all plastic washes into the oceans from land, carried by rivers, floods, or storms, or dumped by maritime vessels. Some of this material becomes trapped in Arctic ice and some, landing on beaches, can even turn into rocks made of plastic. But the vast majority should still be floating out there in the sea, trapped in midocean gyres—large eddies in the center of oceans, like theGreat Pacific Garbage Patch.
To figure out how much refuse is floating in those garbage patches, four ships of the Malaspina expedition, a global research project studying the oceans, fished for plastic across all five major ocean gyres in 2010 and 2011. After months of trailing fine mesh nets around the world, the vessels came up light—by a lot. Instead of the millions of tons scientists had expected, the researchers calculated the global load of ocean plastic to be about only 40,000 tons at the most, the researchers report online today in the Proceedings of the National Academy of Sciences. “We can’t account for 99% of the plastic that we have in the ocean,” says Duarte, the team’s leader.
He suspects that a lot of the missing plastic has been eaten by marine animals. When plastic is floating out on the open ocean, waves and radiation from the sun can fragment it into smaller and smaller particles, until it gets so small it begins to look like fish food—especially to small lanternfish, a widespread small marine fish known to ingest plastic.
“Yes, animals are eating it,” says oceanographer Peter Davison of the Farallon Institute for Advanced Ecosystem Research in Petaluma, California, who was not involved in the study. “That much is indisputable.”
But, he says, it’s hard to know at this time what the biological consequences are. Toxic ocean pollutants like DDT, PCBs, or mercury cling to the surface of plastics, causing them to “suck up all the pollutants in the water and concentrate them.” When animals eat the plastic, that poison could be going into the fish and traveling up the food chain to market species like tuna or swordfish. Or, Davison says, toxins in the fish “may dissolve back into the water … or for all we know they’re puking [the plastic] or pooping it out, and there’s no long-term damage. We just don’t know.”
The last several years have witnessed an explosion in the understanding and use of novel, versatile trans-acting elements. TALEs, CRISPR/Cas, and sRNAs can be easily fashioned to bind any specific sequence of DNA (TALEs, CRISPR/Cas) or RNA (sRNAs) because of the simple rules governing their interactions with nucleic acids. This unique property enables these tools to repress the expression of genes at the transcriptional or post-transcriptional levels, respectively, without prior manipulation of cis-acting and/or chromosomal target DNA sequences. These tools are now being harnessed by synthetic biologists, particularly those in the eukaryotic community, for genome-wide regulation, editing, or epigenetic studies. Here we discuss the exciting opportunities for using TALEs, CRISPR/Cas, and sRNAs as synthetic trans-acting regulators in prokaryotes.