Easy Leaf Area: Automated digital image analysis for rapid and accurate measurement of leaf area.

UnlabelledPremise of the studyMeasurement of leaf areas from digital photographs has traditionally required significant user input unless backgrounds are carefully masked. Easy Leaf Area was developed to batch process hundreds of Arabidopsis rosette images in minutes, removing background artifacts and saving results to a spreadsheet-ready CSV file. •Methods and resultsEasy Leaf Area uses the color ratios of each pixel to distinguish leaves and calibration areas from their background and compares leaf pixel counts to a red calibration area to eliminate the need for camera distance calculations or manual ruler scale measurement that other software methods typically require. Leaf areas estimated by this software from images taken with a camera phone were more accurate than ImageJ estimates from flatbed scanner images. •ConclusionsEasy Leaf Area provides an easy-to-use method for rapid measurement of leaf area and nondestructive estimation of canopy area from digital images

The effects of rising atmospheric carbon dioxide on shoot-root nitrogen and water signaling.

Terrestrial higher plants are composed of roots and shoots, distinct organs that conduct complementary functions in dissimilar environments. For example, roots are responsible for acquiring water and nutrients such as inorganic nitrogen from the soil, yet shoots consume the majority of these resources. The success of such a relationship depends on excellent root-shoot communications. Increased net photosynthesis and decreased shoot nitrogen and water use at elevated CO2 fundamentally alter these source-sink relations. Lower than predicted productivity gains at elevated CO2 under nitrogen or water stress may indicate shoot-root signaling lacks plasticity to respond to rising atmospheric CO2 concentrations. The following presents recent research results on shoot-root nitrogen and water signaling, emphasizing the influence that rising atmospheric carbon dioxide levels are having on these source-sink interactions

Nitrate photo-assimilation in tomato leaves under short-term exposure to elevated carbon dioxide and low oxygen

The role of photorespiration in the foliar assimilation of nitrate (NO 3 -) and carbon dioxide (CO2) was investigated by measuring net CO2 assimilation, net oxygen (O2) evolution, and chlorophyll fluorescence in tomato leaves (Lycopersicon esculentum). The plants were grown under ambient CO2 with ammonium nitrate (NH4NO3) as the nitrogen source, and then exposed to a CO2 concentration of either 360 or 700 μmol mol -1, an O2 concentration of 21 or 2%, and either NO 3 - or NH4 + as the sole nitrogen source. The elevated CO2 concentration stimulated net CO2 assimilation under 21% O2 for both nitrogen treatments, but not under 2% O2. Under ambient CO2 and O2 conditions (i.e. 360 μmol mol-1 CO2, 21% O 2), plants that received NO3 - had 11-13% higher rates of net O2 evolution and electron transport rate (estimated from chlorophyll fluorescence) than plants that received NH 4 +. Differences in net O2 evolution and electron transport rate due to the nitrogen source were not observed at the elevated CO2 concentration for the 21% O2 treatment or at either CO2 level for the 2% O2 treatment. The assimilatory quotient (AQ) from gas exchange, the ratio of net CO2 assimilation to net O2 evolution, indicated more NO3 - - assimilation under ambient CO2 and O 2 conditions than under the other treatments. When the AQ was derived from gross O2 evolution rates estimated from chlorophyll fluorescence, no differences could be detected between the nitrogen treatments. The results suggest that short-term exposure to elevated atmospheric CO 2 decreases NO3 - assimilation in tomato, and that photorespiration may help to support NO3 - assimilation.Fil: Searles, Peter Stoughton. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Regional de Investigaciones Científicas y Transferencia Tecnológica de La Rioja. - Universidad Nacional de La Rioja. Centro Regional de Investigaciones Científicas y Transferencia Tecnológica de La Rioja. - Universidad Nacional de Catamarca. Centro Regional de Investigaciones Científicas y Transferencia Tecnológica de La Rioja. - Secretaría de Industria y Minería. Servicio Geológico Minero Argentino. Centro Regional de Investigaciones Científicas y Transferencia Tecnológica de La Rioja. - Provincia de La Rioja. Centro Regional de Investigaciones Científicas y Transferencia Tecnológica de La Rioja; ArgentinaFil: Bloom, Arnold J.. University of California at Davis; Estados Unido

Nitrate reductase 15N discrimination in Arabidopsis thaliana, Zea mays, Aspergillus niger, Pichea angusta, and Escherichia coli

Stable 15N isotopes have been used to examine movement of nitrogen (N) through various pools of the global N cycle. A central reaction in the cycle involves nitrate (NO3–) reduction to nitrite (NO2–) catalyzed via nitrate reductase (NR). Discrimination against 15N by NR is a major determinant of isotopic differences among N pools. Here, we measured in vitro 15N discrimination by several NRs purified from plants, fungi, and a bacterium to determine the intrinsic 15N discrimination by the enzyme and to evaluate the validity of measurements made using 15N-enriched NO3–. Observed NR isotope discrimination ranged from 22‰ to 32‰ (kinetic isotope effects of 1.022 to 1.032) among the different isozymes at natural abundance 15N (0.37%). As the fractional 15N content of substrate NO3– increased from natural abundance, the product 15N fraction deviated significantly from that expected based on substrate enrichment and 15N discrimination measured at natural abundance. Additionally, isotopic discrimination by denitrifying bacteria used to reduce NO3– and NO2– in some protocols became a greater source of error as 15N enrichment increased. We briefly discuss potential causes of artifactual results with enriched 15N and recommend against the use of highly enriched 15N tracers to study N discrimination in plants or soils

Deposition of ammonium and nitrate in the roots of maize seedlings supplied with different nitrogen salts

This study measured total osmolarity and concentrations of NH4+, NO3–, K+, soluble carbohydrates, and organic acids in maize seminal roots as a function of distance from the apex, and NH4+ and NO3– in xylem sap for plants receiving NH4+ or NO3– as a sole N-source, NH4+ plus NO3–, or no nitrogen at all. The disparity between net deposition rates and net exogenous influx of NH4+ indicated that growing cells imported NH4+ from more mature tissue, whereas more mature root tissues assimilated or translocated a portion of the NH4+ absorbed. Net root NO3– influx under Ca(NO3)2 nutrition was adequate to account for pools found in the growth zone and provided twice as much as was deposited locally throughout the non-growing tissue. In contrast, net root NO3– influx under NH4NO3 was less than the local deposition rate in the growth zone, indicating that additional NO3– was imported or metabolically produced. The profile of NO3– deposition rate in the growth zone, however, was similar for the plants receiving Ca(NO3)2 or NH4NO3. These results suggest that NO3– may serve a major role as an osmoticant for supporting root elongation in the basal part of the growth zone and maintaining root function in the young mature tissues

In the light of directed evolution: Pathways of adaptive protein evolution

Directed evolution is a widely-used engineering strategy for improving the stabilities or biochemical functions of proteins by repeated rounds of mutation and selection. These experiments offer empirical lessons about how proteins evolve in the face of clearly-defined laboratory selection pressures. Directed evolution has revealed that single amino acid mutations can enhance properties such as catalytic activity or stability and that adaptation can often occur through pathways consisting of sequential beneficial mutations. When there are no single mutations that improve a particular protein property experiments always find a wealth of mutations that are neutral with respect to the laboratory-defined measure of fitness. These neutral mutations can open new adaptive pathways by at least 2 different mechanisms. Functionally-neutral mutations can enhance a protein's stability, thereby increasing its tolerance for subsequent functionally beneficial but destabilizing mutations. They can also lead to changes in “promiscuous” functions that are not currently under selective pressure, but can subsequently become the starting points for the adaptive evolution of new functions. These lessons about the coupling between adaptive and neutral protein evolution in the laboratory offer insight into the evolution of proteins in nature

Why highly expressed proteins evolve slowly

Much recent work has explored molecular and population-genetic constraints on the rate of protein sequence evolution. The best predictor of evolutionary rate is expression level, for reasons which have remained unexplained. Here, we hypothesize that selection to reduce the burden of protein misfolding will favor protein sequences with increased robustness to translational missense errors. Pressure for translational robustness increases with expression level and constrains sequence evolution. Using several sequenced yeast genomes, global expression and protein abundance data, and sets of paralogs traceable to an ancient whole-genome duplication in yeast, we rule out several confounding effects and show that expression level explains roughly half the variation in Saccharomyces cerevisiae protein evolutionary rates. We examine causes for expression's dominant role and find that genome-wide tests favor the translational robustness explanation over existing hypotheses that invoke constraints on function or translational efficiency. Our results suggest that proteins evolve at rates largely unrelated to their functions, and can explain why highly expressed proteins evolve slowly across the tree of life.Comment: 40 pages, 3 figures, with supporting informatio

Impacts of elevated atmospheric CO₂ on nutrient content of important food crops.

One of the many ways that climate change may affect human health is by altering the nutrient content of food crops. However, previous attempts to study the effects of increased atmospheric CO2 on crop nutrition have been limited by small sample sizes and/or artificial growing conditions. Here we present data from a meta-analysis of the nutritional contents of the edible portions of 41 cultivars of six major crop species grown using free-air CO2 enrichment (FACE) technology to expose crops to ambient and elevated CO2 concentrations in otherwise normal field cultivation conditions. This data, collected across three continents, represents over ten times more data on the nutrient content of crops grown in FACE experiments than was previously available. We expect it to be deeply useful to future studies, such as efforts to understand the impacts of elevated atmospheric CO2 on crop macro- and micronutrient concentrations, or attempts to alleviate harmful effects of these changes for the billions of people who depend on these crops for essential nutrients

Inclusive Electron Scattering from Nuclei at x≃1x \simeq 1

The inclusive A(e,e') cross section for $x \simeq 1$ was measured on $^2$H, C, Fe, and Au for momentum transfers $Q^2$ from 1-7 (GeV/c)$^2$. The scaling behavior of the data was examined in the region of transition from y-scaling to x-scaling. Throughout this transitional region, the data exhibit $\xi$-scaling, reminiscent of the Bloom-Gilman duality seen in free nucleon scattering.Comment: 4 pages, RevTeX; 4 figures (postscript in .tar.Z file

Expanded molecular diversity generation during directed evolution by trinucleotide exchange (TriNEx)

Trinucleotide exchange (TriNEx) is a method for generating novel molecular diversity during directed evolution by random substitution of one contiguous trinucleotide sequence for another. Single trinucleotide sequences were deleted at random positions in a target gene using the engineered transposon MuDel that were subsequently replaced with a randomized trinucleotide sequence donated by the DNA cassette termed SubSeqNNN. The bla gene encoding TEM-1 β-lactamase was used as a model to demonstrate the effectiveness of TriNEx. Sequence analysis revealed that the mutations were distributed throughout bla, with variants containing single, double and triple nucleotide changes. Many of the resulting amino acid substitutions had significant effects on the in vivo activity of TEM-1, including up to a 64-fold increased activity toward ceftazidime and up to an 8-fold increased resistance to the inhibitor clavulanate. Many of the observed amino acid substitutions were only accessible by exchanging at least two nucleotides per codon, including charge-switch (R164D) and aromatic substitution (W165Y) mutations. TriNEx can therefore generate a diverse range of protein variants with altered properties by combining the power of site-directed saturation mutagenesis with the capacity of whole-gene mutagenesis to randomly introduce mutations throughout a gene