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

Neutral genetic drift can aid functional protein evolution

BACKGROUND: Many of the mutations accumulated by naturally evolving proteins are neutral in the sense that they do not significantly alter a protein's ability to perform its primary biological function. However, new protein functions evolve when selection begins to favor other, "promiscuous" functions that are incidental to a protein's biological role. If mutations that are neutral with respect to a protein's primary biological function cause substantial changes in promiscuous functions, these mutations could enable future functional evolution. RESULTS: Here we investigate this possibility experimentally by examining how cytochrome P450 enzymes that have evolved neutrally with respect to activity on a single substrate have changed in their abilities to catalyze reactions on five other substrates. We find that the enzymes have sometimes changed as much as four-fold in the promiscuous activities. The changes in promiscuous activities tend to increase with the number of mutations, and can be largely rationalized in terms of the chemical structures of the substrates. The activities on chemically similar substrates tend to change in a coordinated fashion, potentially providing a route for systematically predicting the change in one function based on the measurement of several others. CONCLUSIONS: Our work suggests that initially neutral genetic drift can lead to substantial changes in protein functions that are not currently under selection, in effect poising the proteins to more readily undergo functional evolution should selection "ask new questions" in the future

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

Structure-Guided Recombination Creates an Artificial Family of Cytochromes P450

Creating artificial protein families affords new opportunities to explore the determinants of structure and biological function free from many of the constraints of natural selection. We have created an artificial family comprising ~3,000 P450 heme proteins that correctly fold and incorporate a heme cofactor by recombining three cytochromes P450 at seven crossover locations chosen to minimize structural disruption. Members of this protein family differ from any known sequence at an average of 72 and by as many as 109 amino acids. Most (>73%) of the properly folded chimeric P450 heme proteins are catalytically active peroxygenases; some are more thermostable than the parent proteins. A multiple sequence alignment of 955 chimeras, including both folded and not, is a valuable resource for sequence-structure-function studies. Logistic regression analysis of the multiple sequence alignment identifies key structural contributions to cytochrome P450 heme incorporation and peroxygenase activity and suggests possible structural differences between parents CYP102A1 and CYP102A2

The Effects of Inorganic Nitrogen form and CO2CO_2 Concentration on Wheat Yield and Nutrient Accumulation and Distribution

Inorganic N is available to plants from the soil as ammonium ($NH^+_4$) and nitrate ($NO^-_3$). We studied how wheat grown hydroponically to senescence in controlled environmental chambers is affected by N form ($NH^+_4$ vs. $NO^-_3$) and $CO_2$ concentration (“subambient,” “ambient,” and “elevated”) in terms of biomass, yield, and nutrient accumulation and partitioning. Wheat supplied with $NH^+_4$ as a sole N source had the strongest response to $CO_2$ concentration. Plants exposed to subambient and ambient $CO_2$ concentrations typically had the greatest biomass and nutrient accumulation under both N forms. In general $NH^+_4$-supplied plants had higher concentrations of total N, P, K, S, Ca, Zn, Fe, and Cu, while $NO^-_3$-supplied plants had higher concentrations of Mg, B, Mn, and $NO^-_3$ - N. $NH^+_4$-supplied plants contained amounts of phytate similar to $NO^-_3$-supplied plants but had higher bioavailable Zn, which could have consequences for human health. $NH^+_4$-supplied plants allocated more nutrients and biomass to aboveground tissues whereas $NO^+_3$-supplied plants allocated more nutrients to the roots. The two inorganic nitrogen forms influenced plant growth and nutrient status so distinctly that they should be treated as separate nutrients. Moreover, plant growth and nutrient status varied in a non-linear manner with atmospheric $CO_2$ concentration

Breaking proteins with mutations: threads and thresholds in evolution

A common high school science experiment involves anchoring one end of a rubber band to a desk and then attaching a small weight to the other end. The weight stretches the rubber band, and adding another weight causes the rubber band to dangle even lower. More weights can be added, and each one pulls the rubber band a little further towards the floor. Now, instead imagine attaching the weights to a thread. The thread stretches only slightly; so the first couple of weights have just a small effect. But if you add enough weights, the thread suddenly breaks and the weights fall to the floor. In the first case, each additional weight stretches the rubber band by the same amount, whereas in the second, it is the combination of several weights that breaks the thread. Mutating proteins is like adding weights, as mutations eventually ‘break’ the individual proteins, dragging down the fraction of proteins that still function (this fraction is the average fitness)

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

Consensus Protein Design without Phylogenetic Bias

Consensus design is an appealing strategy for the stabilization of proteins. It exploits amino acid conservation in sets of homologous proteins to identify likely beneficial mutations. Nevertheless, its success depends on the phylogenetic diversity of the sequence set available. Here, we show that randomization of a single protein represents a reliable alternative source of sequence diversity that is essentially free of phylogenetic bias. A small number of functional protein sequences selected from binary-patterned libraries suffice as input for the consensus design of active enzymes that are easier to produce and substantially more stable than individual members of the starting data set. Although catalytic activity correlates less consistently with sequence conservation in these extensively randomized proteins, less extreme mutagenesis strategies might be adopted in practice to augment stability while maintaining function