The lion's share of bacteria in various environments cannot be cloned in the laboratory and thus cannot be sequenced using existing technologies. A major goal of single-cell genomics is to complement gene-centric metagenomic data with whole-genome assemblies of uncultivated organisms. Assembly of single-cell data is challenging because of highly non-uniform read coverage as well as elevated levels of sequencing errors and chimeric reads. We describe SPAdes, a new assembler for both single-cell and standard (multicell) assembly, and demonstrate that it improves on the recently released E+V-SC assembler (specialized for single-cell data) and on popular assemblers Velvet and SoapDeNovo (for multicell data). SPAdes generates single-cell assemblies, providing information about genomes of uncultivatable bacteria that vastly exceeds what may be obtained via traditional metagenomics studies. SPAdes is available online ( http://bioinf.spbau.ru/spades ). It is distributed as open source software.

SPAdes, a new genome assembly algorithm and its applications to single-cell sequencing ( 7th Annual SFAF Meeting, 2012)

Improving plant productivity is an important aim for metabolic engineering. There are few comprehensive methods that quantitatively describe leaf metabolism, although such information would be valuable for increasing photosynthetic capacity, enhancing biomass production, and rerouting carbon flux toward desirable end products. Isotopically nonstationary metabolic flux analysis (INST-MFA) has been previously applied to map carbon fluxes in photoautotrophic bacteria, which involves model-based regression of transient 13C-labeling patterns of intracellular metabolites. However, experimental and computational difficulties have hindered its application to terrestrial plant systems. We performed in vivo isotopic labeling of Arabidopsis thaliana rosettes with 13CO2 and estimated fluxes throughout leaf photosynthetic metabolism by INST-MFA. Plants grown at 200 µmol m-2s−1 light were compared with plants acclimated for 9 d at an irradiance of 500 µmol⋅m−2⋅s−1. Approximately 1,400 independent mass isotopomer measurements obtained from analysis of 37 metabolite fragment ions were regressed to estimate 136 total fluxes (54 free fluxes) under each condition. The results provide a comprehensive description of changes in carbon partitioning and overall photosynthetic flux after long-term developmental acclimation of leaves to high light. Despite a doubling in the carboxylation rate, the photorespiratory flux increased from 17 to 28% of net CO2 assimilation with high-light acclimation (Vc/Vo: 3.5:1 vs. 2.3:1, respectively). This study highlights the potential of 13C INST-MFA to describe emergent flux phenotypes that respond to environmental conditions or plant physiology and cannot be obtained by other complementary approaches.

/pdf/isotopically-nonstationary-13c-flux-analysis-of-changes-in-50wxb22shu.pdf

Isotopically nonstationary 13C flux analysis of changes in Arabidopsis thaliana leaf metabolism due to high light acclimation.

Although leaves have to accommodate markedly different metabolic flux patterns in the light and the dark, models of leaf metabolism based on flux-balance analysis (FBA) have so far been confined to consideration of the network under continuous light. An FBA framework is presented that solves the two phases of the diel cycle as a single optimization problem and, thus, provides a more representative model of leaf metabolism. The requirement to support continued export of sugar and amino acids from the leaf during the night and to meet overnight cellular maintenance costs forces the model to set aside stores of both carbon and nitrogen during the day. With only minimal constraints, the model successfully captures many of the known features of C3 leaf metabolism, including the recently discovered role of citrate synthesis and accumulation in the night as a precursor for the provision of carbon skeletons for amino acid synthesis during the day. The diel FBA model can be applied to other temporal separations, such as that which occurs in Crassulacean acid metabolism (CAM) photosynthesis, allowing a system-level analysis of the energetics of CAM. The diel model predicts that there is no overall energetic advantage to CAM, despite the potential for suppression of photorespiration through CO2 concentration. Moreover, any savings in enzyme machinery costs through suppression of photorespiration are likely to be offset by the higher flux demand of the CAM cycle. It is concluded that energetic or nitrogen use considerations are unlikely to be evolutionary drivers for CAM photosynthesis.

/pdf/a-diel-flux-balance-model-captures-interactions-between-4h094u7gae.pdf

A Diel Flux Balance Model Captures Interactions between Light and Dark Metabolism during Day-Night Cycles in C3 and Crassulacean Acid Metabolism Leaves.

This article reviews recent literature on drought of the last millennium, followed by an update on global aridity changes from 1950 to 2008. Projected future aridity is presented based on recent studies and our analysis of model simulations. Dry periods lasting for years to decades have occurred many times during the last millennium over, for example, North America, West Africa, and East Asia. These droughts were likely triggered by anomalous tropical sea surface temperatures (SSTs), with La Ni˜ na-like SST anomalies leading to drought in North America, and El-Ni˜ no-like SSTs causing drought in East China. Over Africa, the southward shift of the warmest SSTs in the Atlantic and warming in the Indian Ocean are responsible for the recent Sahel droughts. Local feedbacks may enhance and prolong drought. Global aridity has increased substantially since the 1970s due to recent drying over Africa, southern Europe, East and South Asia, and eastern Australia. Although El Ni˜ no-Southern Oscillation (ENSO), tropical Atlantic SSTs, and Asian monsoons have played a large role in the recent drying, recent warming has increased atmospheric moisture demand and likely altered atmospheric circulation patterns, both contributing to the drying. Climate models project increased aridity in the 21 st century over most of Africa, southern Europe and the Middle East, most of the Americas, Australia, and Southeast Asia. Regions like the United States have avoided prolonged droughts during the last 50 years due to natural climate variations, but might see persistent droughts in the next 20–50 years. Future efforts to predict drought will depend on models’ ability to predict tropical SSTs. 2010 JohnWiley &Sons,Ltd.WIREs Clim Change2010 DOI:10.1002/wcc.81

Drought under Global Warming: A Review

Protein turnover is a key component in cellular homeostasis; however, there is little quantitative information on degradation kinetics for individual plant proteins. We have used 15N labeling of barley (Hordeum vulgare) plants and gas chromatography-mass spectrometry analysis of free amino acids and liquid chromatography-mass spectrometry analysis of proteins to track the enrichment of 15N into the amino acid pools in barley leaves and then into tryptic peptides derived from newly synthesized proteins. Using information on the rate of growth of barley leaves combined with the rate of degradation of 14N-labeled proteins, we calculate the turnover rates of 508 different proteins in barley and show that they vary by more than 100-fold. There was approximately a 9-h lag from label application until 15N incorporation could be reliably quantified in extracted peptides. Using this information and assuming constant translation rates for proteins during the time course, we were able to quantify degradation rates for several proteins that exhibit half-lives on the order of hours. Our workflow, involving a stringent series of mass spectrometry filtering steps, demonstrates that 15N labeling can be used for large-scale liquid chromatography-mass spectrometry studies of protein turnover in plants. We identify a series of abundant proteins in photosynthesis, photorespiration, and specific subunits of chlorophyll biosynthesis that turn over significantly more rapidly than the average protein involved in these processes. We also highlight a series of proteins that turn over as rapidly as the well-known D1 subunit of photosystem II. While these proteins need further verification for rapid degradation in vivo, they cluster in chlorophyll and thiamine biosynthesis.

/pdf/proteins-with-high-turnover-rate-in-barley-leaves-estimated-bzlee33knc.pdf

Proteins with High Turnover Rate in Barley Leaves Estimated by Proteome Analysis Combined with in Planta Isotope Labeling

We describe the construction and analysis of a genome-scale metabolic model representing a developing leaf cell of rice (Oryza sativa) primarily derived from the annotations in the RiceCyc database. We used flux balance analysis to determine that the model represents a network capable of producing biomass precursors (amino acids, nucleotides, lipid, starch, cellulose, and lignin) in experimentally reported proportions, using carbon dioxide as the sole carbon source. We then repeated the analysis over a range of photon flux values to examine responses in the solutions. The resulting flux distributions show that (1) redox shuttles between the chloroplast, cytosol, and mitochondrion may play a significant role at low light levels, (2) photorespiration can act to dissipate excess energy at high light levels, and (3) the role of mitochondrial metabolism is likely to vary considerably according to the balance between energy demand and availability. It is notable that these organelle interactions, consistent with many experimental observations, arise solely as a result of the need for mass and energy balancing without any explicit assumptions concerning kinetic or other regulatory mechanisms.

/pdf/responses-to-light-intensity-in-a-genome-scale-model-of-rice-4zl2kvkw27.pdf

Responses to Light Intensity in a Genome-Scale Model of Rice Metabolism

Single-Cell Transcriptome Analysis in Plants: Advances and Challenges

Responses to Light Intensity in a Genome-Scale Model of Rice Metabolism1[C][W][OA]

Plant metabolism is highly adapted in response to its surrounding for acquiring limiting resources. In this study, a dynamic flux balance modeling framework with a multi-tissue (leaf and root) diel genome-scale metabolic model of Arabidopsis thaliana was developed and applied to investigate the reprogramming of plant metabolism through multiple growth stages under different nutrient availability. The framework allowed the modeling of optimal partitioning of resources and biomass in leaf and root over diel phases. A qualitative flux map of carbon and nitrogen metabolism was identified which was consistent across growth phases under both nitrogen rich and limiting conditions. Results from the model simulations suggested distinct metabolic roles in nitrogen metabolism played by enzymes with different cofactor specificities. Moreover, the dynamic model was used to predict the effect of physiological or environmental perturbation on the growth of Arabidopsis leaves and roots.

/pdf/a-dynamic-multi-tissue-flux-balance-model-captures-carbon-20k7v3jqce.pdf

A Dynamic Multi-Tissue Flux Balance Model Captures Carbon and Nitrogen Metabolism and Optimal Resource Partitioning During Arabidopsis Growth.

Until they become photoautotrophic juvenile plants, seedlings depend upon the reserves stored in seed tissues. These reserves must be mobilized and metabolized, and their breakdown products must be distributed to the different organs of the growing seedling. Here, we investigated the mobilization of soybean (Glycine max) seed reserves during seedling growth by initially constructing a genome-scale stoichiometric model for this important crop plant and then adapting the model to reflect metabolism in the cotyledons and hypocotyl/root axis (HRA). A detailed analysis of seedling growth and alterations in biomass composition was performed over 4 d of postgerminative growth and used to constrain the stoichiometric model. Flux balance analysis revealed marked differences in metabolism between the two organs, together with shifts in primary metabolism occurring during different periods postgermination. In particular, from 48 h onward, cotyledons were characterized by the oxidation of fatty acids to supply carbon for the tricarboxylic acid cycle as well as production of sucrose and glutamate for export to the HRA, while the HRA was characterized by the use of a range of imported amino acids in protein synthesis and catabolic processes. Overall, the use of flux balance modeling provided new insight into well-characterized metabolic processes in an important crop plant due to their analysis within the context of a metabolic network and reinforces the relevance of the application of this technique to the analysis of complex plant metabolic systems.

/pdf/a-genome-scale-metabolic-model-of-soybean-glycine-max-329yyhze5p.pdf

Rahul Shaw

Papers

Responses to Light Intensity in a Genome-Scale Model of Rice Metabolism

Single-Cell Transcriptome Analysis in Plants: Advances and Challenges

Responses to Light Intensity in a Genome-Scale Model of Rice Metabolism1[C][W][OA]

A Dynamic Multi-Tissue Flux Balance Model Captures Carbon and Nitrogen Metabolism and Optimal Resource Partitioning During Arabidopsis Growth.

A Genome-Scale Metabolic Model of Soybean ( Glycine max) Highlights Metabolic Fluxes in Seedlings