Novel method for the quantification of rosette area from images of Arabidopsis seedlings grown on agar plates.

Premise: The agar-based culture of Arabidopsis seedlings is widely used for quantifying root traits. Shoot traits are generally overlooked in these studies, probably because the rosettes are often askew. A technique to assess the shoot surface area of seedlings grown inside agar culture dishes would facilitate simultaneous root and shoot phenotyping. Methods: We developed an image processing workflow in Python that estimates rosette area of Arabidopsis seedlings on agar culture dishes. We validated this method by comparing its output with other metrics of seedling growth. As part of a larger study on genetic variation in plant responses to nitrogen form and concentration, we measured the rosette areas from more than 2000 plate images. Results: The rosette area measured from plate images was strongly correlated with the rosette area measured from directly overhead and moderately correlated with seedling mass. Rosette area in the large image set was significantly influenced by genotype and nitrogen treatment. The broad-sense heritability of leaf area measured using this method was 0.28. Discussion: These results indicated that this approach for estimating rosette area produces accurate shoot phenotype data. It can be used with image sets for which other methods of leaf area quantification prove unsuitable.

Genetic variation underlying differential ammonium and nitrate responses in Arabidopsis thaliana.

Nitrogen is an essential element required for plant growth and productivity. Understanding the mechanisms and natural genetic variation underlying nitrogen use in plants will facilitate the engineering of plant nitrogen use to maximize crop productivity while minimizing environmental costs. To understand the scope of natural variation that may influence nitrogen use, we grew 1,135 Arabidopsis thaliana natural genotypes on two nitrogen sources, nitrate and ammonium, and measured both developmental and defense metabolite traits. By using different environments and focusing on multiple traits, we identified a wide array of different nitrogen responses. These responses are associated with numerous genes, most of which were not previously associated with nitrogen responses. Only a small portion of these genes appear to be shared between environments or traits, while most are predominantly specific to a developmental or defense trait under a specific nitrogen source. Finally, by using a large population, we were able to identify unique nitrogen responses, such as preferring ammonium or nitrate, which appear to be generated by combinations of loci rather than a few large-effect loci. This suggests that it may be possible to obtain novel phenotypes in complex nitrogen responses by manipulating sets of genes with small effects rather than solely focusing on large-effect single gene manipulations.

Remote learning slightly decreased student performance in an introductory undergraduate course on climate change.

Public understanding about complex issues such as climate change relies heavily on online resources. Yet the role that online instruction should assume in post-secondary science education remains contentious despite its near ubiquity during the COVID-19 pandemic. The objective here was to compare the performance of 1790 undergraduates taking either an online or face-to-face version of an introductory course on climate change. Both versions were taught by a single instructor, thus, minimizing instructor bias. Women, seniors, English language learners, and humanities majors disproportionately chose to enroll in the online version because of its ease of scheduling and accessibility. After correcting for performance-gaps among different demographic groups, the COVID-19 pandemic had no significant effect on online student performance and students in the online version scored 2% lower (on a scale of 0-100) than those in the face-to-face version, a penalty that may be a reasonable tradeoff for the ease of scheduling and accessibility that these students desire.

Easy as piadcs: A low-cost, ultra-high-resolution data acquisition system using a Raspberry Pi.

Premise: High-precision data acquisition (DAQ) is essential for developing new methods in the plant sciences. Commercial high-resolution DAQ systems are cost prohibitive, whereas the less expensive systems that are currently available lack the resolution and precision required for many physiological measurements. Methods and Results: We developed the software libraries, called piadcs, and hardware design for a DAQ system based on an ultra-high-resolution analog-to-digital converter and a Raspberry Pi computer. We tested the system precision with and without a thermocouple attached and found the precision with the sensor to be better than ±0.01°C and the maximum possible system resolution to be 0.4 ppm. Conclusions: The ultra-high-resolution DAQ system described here is inexpensive, flexible enough to be used with many different sensors, and can be built by researchers with rudimentary electronic and computer skills. This system is most applicable in the development of new measurement techniques and the improvement of existing methods.

Breeding for Higher Yields of Wheat and Rice through Modifying Nitrogen Metabolism.

Wheat and rice produce nutritious grains that provide 32% of the protein in the human diet globally. Here, we examine how genetic modifications to improve assimilation of the inorganic nitrogen forms ammonium and nitrate into protein influence grain yield of these crops. Successful breeding for modified nitrogen metabolism has focused on genes that coordinate nitrogen and carbon metabolism, including those that regulate tillering, heading date, and ammonium assimilation. Gaps in our current understanding include (1) species differences among candidate genes in nitrogen metabolism pathways, (2) the extent to which relative abundance of these nitrogen forms across natural soil environments shape crop responses, and (3) natural variation and genetic architecture of nitrogen-mediated yield improvement. Despite extensive research on the genetics of nitrogen metabolism since the rise of synthetic fertilizers, only a few projects targeting nitrogen pathways have resulted in development of cultivars with higher yields. To continue improving grain yield and quality, breeding strategies need to focus concurrently on both carbon and nitrogen assimilation and consider manipulating genes with smaller effects or that underlie regulatory networks as well as genes directly associated with nitrogen metabolism.

Wheat grain yield decreased over the past 35 years, but protein content did not change.

The extent to which rising atmospheric CO2 concentration has already influenced food production and quality is uncertain. Here, we analyzed annual field trials of autumn-planted common wheat in California from 1985 to 2019, a period during which the global atmospheric CO2 concentration increased 19%. Even after accounting for other major factors (cultivar, location, degree-days, soil temperature, total water applied, nitrogen fertilization, and pathogen infestation), wheat grain yield and protein yield declined 13% over this period, but grain protein content did not change. These results suggest that exposure to gradual CO2 enrichment over the past 35 years has adversely affected wheat grain and protein yield, but not grain protein content.

Rising atmospheric CO2 concentration inhibits nitrate assimilation in shoots but enhances it in roots of C3 plants.

We have proposed that rising atmospheric CO2 concentrations inhibit malate production in chloroplasts and thus impede assimilation of nitrate into protein in shoots of C3 plants, a phenomenon that will strongly influence primary productivity and food security under the environmental conditions anticipated during the next few decades. Although hundreds of studies support this proposal, several publications in 2018 and 2019 purport to present counterevidence. The following study evaluates these publications as well as presents new data that elevated CO2 enhances root nitrate assimilation in wheat and Arabidopsis while it inhibits shoot nitrate assimilation.

Author Correction: Increasing CO2 threatens human nutrition.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Metal regulation of metabolism.

A broad range of biochemicals, from proteins to nucleic acids, function properly only when associated with a metal, usually a divalent cation. Not any divalent metal will do: these metals differ in their ionic radius, dissociation in water, ionization potential, and number of unpaired electrons in their outer shells, and so substituting one metal for another often changes substrate positioning, redox reactivities, and physiological performance, and thus may serve as a regulatory mechanism. For instance, exchanging manganese for magnesium in several chloroplast enzymes maintains plant carbon-nitrogen balance under rising atmospheric CO2 concentrations. Here, we review this and a few other cases where association of proteins or nucleic acids with different metals control metabolism.

Manganese binding to Rubisco could drive a photorespiratory pathway that increases the energy efficiency of photosynthesis.

Most plants, contrary to popular belief, do not waste over 30% of their photosynthate in a futile cycle called photorespiration. Rather, the photorespiratory pathway generates additional malate in the chloroplast that empowers many energy-intensive chemical reactions, such as those involved in nitrate assimilation. Thus, the balance between carbon fixation and photorespiration determines the plant carbon-nitrogen balance and protein concentrations. Plant protein concentrations, in turn, depend not only on the relative concentrations of carbon dioxide and oxygen in the chloroplast but also on the relative activities of magnesium and manganese, which are metals that associate with several key enzymes in the photorespiratory pathway and alter their function. Understanding the regulation of these processes is critical for sustaining food quality under rising CO2 atmospheres.

Relative association of Rubisco with manganese and magnesium as a regulatory mechanism in plants.

Rubisco, the enzyme that constitutes as much as half of the protein in a leaf, initiates either the photorespiratory pathway that supplies reductant for the assimilation of nitrate into amino acids or the C3 carbon fixation pathway that generates carbohydrates. The relative rates of these two pathways depend both on the relative extent to which O2 and CO2 occupies the active site of Rubisco and on whether manganese or magnesium is bound to the enzyme. This study quantified the activities of manganese and magnesium in isolated tobacco chloroplasts and the thermodynamics of binding of these metals to Rubisco purified from tobacco or a bacterium. In tobacco chloroplasts, manganese was less active than magnesium, but Rubisco purified from tobacco had a higher affinity for manganese. The activity of each metal in the chloroplast was similar in magnitude to the affinity of tobacco Rubisco for each. This indicates that, in tobacco chloroplasts, Rubisco associates almost equally with both metals and rapidly exchanges one metal for the other. Binding of magnesium was similar in Rubisco from tobacco and a bacterium, whereas binding of manganese differed greatly between the Rubisco from these species. Moreover, the ratio of leaf manganese to magnesium in C3 plants increased as atmospheric CO2 increased. These results suggest that Rubisco has evolved to improve the energy transfers between photorespiration and nitrate assimilation and that plants regulate manganese and magnesium activities in the chloroplast to mitigate detrimental changes in their nitrogen/carbon balance as atmospheric CO2 varies.

Inorganic nitrogen form: a major player in wheat and Arabidopsis responses to elevated CO2.

Critical for predicting the future of primary productivity is a better understanding of plant responses to rising atmospheric carbon dioxide (CO2) concentration. This review considers recent results on the role of the inorganic nitrogen (N) forms nitrate (NO3-) and ammonium (NH4+) in determining the responses of wheat and Arabidopsis to elevated atmospheric CO2 concentration. Here, we identify four key issues: (i) the possibility that different plant species respond similarly to elevated CO2 if one accounts for the N form that they are using; (ii) the major influence that plant-soil N interactions have on plant responses to elevated CO2; (iii) the observation that elevated CO2 may favor the uptake of one N form over others; and (iv) the finding that plants receiving NH4+ nutrition respond more positively to elevated CO2 than those receiving NO3- nutrition because elevated CO2 inhibits the assimilation of NO3- in shoots of C3 plants. We conclude that the form and amount of N available to plants from the rhizosphere and plant preferences for the different N forms are essential for predicting plant responses to elevated CO2.

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.

High-resolution mapping of a major effect QTL from wild tomato Solanum habrochaites that influences water relations under root chilling.

QTL stm9 controlling rapid-onset water stress tolerance in S. habrochaites was high-resolution mapped to a chromosome 9 region that contains genes associated with abiotic stress tolerances. Wild tomato (Solanum habrochaites) exhibits tolerance to abiotic stresses, including drought and chilling. Root chilling (6 °C) induces rapid-onset water stress by impeding water movement from roots to shoots. S. habrochaites responds to such changes by closing stomata and maintaining shoot turgor, while cultivated tomato (S. lycopersicum) fails to close stomata and wilts. This response (shoot turgor maintenance under root chilling) is controlled by a major QTL (designated stm9) on chromosome 9, which was previously fine-mapped to a 2.7-cM region. Recombinant sub-near-isogenic lines for chromosome 9 were marker-selected, phenotyped for shoot turgor maintenance under root chilling in two sets of replicated experiments (Fall and Spring), and the data were used to high-resolution map QTL stm9 to a 0.32-cM region. QTL mapping revealed a single QTL that was coincident for both the Spring and Fall datasets, suggesting that the gene or genes contributing to shoot turgor maintenance under root chilling reside within the marker interval H9-T1673. In the S. lycopersicum reference genome sequence, this chromosome 9 region is gene-rich and contains representatives of gene families that have been associated with abiotic stress tolerance.

The increasing importance of distinguishing among plant nitrogen sources.

Many studies of plant nitrogen relations assess only the total amount of the element available from the soil and the total amount of the element within the plant. Nitrogen, however, is a constituent of diverse compounds that participate in some of the most energy-intensive reactions in the biosphere. The following characterizes some of these reactions, especially those that involve ammonium and nitrate, and highlights the importance of distinguishing both among the nitrogen sources available to plants and among the nitrogen forms within plants when considering plant responses to rising atmospheric CO2 concentrations.

Responses of Arabidopsis and wheat to rising CO2 depend on nitrogen source and nighttime CO2 levels.

A major contributor to the global carbon cycle is plant respiration. Elevated atmospheric CO2 concentrations may either accelerate or decelerate plant respiration for reasons that have been uncertain. We recently established that elevated CO2 during the daytime decreases plant mitochondrial respiration in the light and protein concentration because CO2 slows the daytime conversion of nitrate (NO3 (-)) into protein. This derives in part from the inhibitory effect of CO2 on photorespiration and the dependence of shoot NO3 (-) assimilation on photorespiration. Elevated CO2 also inhibits the translocation of nitrite into the chloroplast, a response that influences shoot NO3 (-) assimilation during both day and night. Here, we exposed Arabidopsis (Arabidopsis thaliana) and wheat (Triticum aestivum) plants to daytime or nighttime elevated CO2 and supplied them with NO3 (-) or ammonium as a sole nitrogen (N) source. Six independent measures (plant biomass, shoot NO3 (-), shoot organic N, (15)N isotope fractionation, (15)NO3 (-) assimilation, and the ratio of shoot CO2 evolution to O2 consumption) indicated that elevated CO2 at night slowed NO3 (-) assimilation and thus decreased dark respiration in the plants reliant on NO3 (-). These results provide a straightforward explanation for the diverse responses of plants to elevated CO2 at night and suggest that soil N source will have an increasing influence on the capacity of plants to mitigate human greenhouse gas emissions.

Does low stomatal conductance or photosynthetic capacity enhance growth at elevated CO2 in Arabidopsis?

The objective of this study was to determine if low stomatal conductance (g) increases growth, nitrate (NO3 (-)) assimilation, and nitrogen (N) utilization at elevated CO2 concentration. Four Arabidopsis (Arabidopsis thaliana) near isogenic lines (NILs) differing in g were grown at ambient and elevated CO2 concentration under low and high NO3 (-) supply as the sole source of N. Although g varied by 32% among NILs at elevated CO2, leaf intercellular CO2 concentration varied by only 4% and genotype had no effect on shoot NO3 (-) concentration in any treatment. Low-g NILs showed the greatest CO2 growth increase under N limitation but had the lowest CO2 growth enhancement under N-sufficient conditions. NILs with the highest and lowest g had similar rates of shoot NO3 (-) assimilation following N deprivation at elevated CO2 concentration. After 5 d of N deprivation, the lowest g NIL had 27% lower maximum carboxylation rate and 23% lower photosynthetic electron transport compared with the highest g NIL. These results suggest that increased growth of low-g NILs under N limitation most likely resulted from more conservative N investment in photosynthetic biochemistry rather than from low g.

Photorespiration and nitrate assimilation: a major intersection between plant carbon and nitrogen.

C3 carbon fixation has a bad reputation, primarily because it is associated with photorespiration, a biochemical pathway thought to waste a substantial amount of the carbohydrate produced in a plant. This review presents evidence collected over nearly a century that (1) Rubisco when associated with Mn(2+) generates additional reductant during photorespiration, (2) this reductant participates in the assimilation of nitrate into protein, and (3) this nitrate assimilation facilitates the use of a nitrogen source that other organisms tend to avoid. This phenomenon explains the continued dominance of C3 plants during the past 23 million years of low CO2 atmospheres as well as the decline in plant protein concentrations as atmospheric CO2 rises.

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

PREMISE OF THE STUDY: Measurement 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 RESULTS: Easy 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. • CONCLUSIONS: Easy Leaf Area provides an easy-to-use method for rapid measurement of leaf area and nondestructive estimation of canopy area from digital images.

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

Stable (15)N 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 the reduction of nitrate (NO(-) 3) to nitrite (NO(-) 2) catalyzed by nitrate reductase (NR). Discrimination against (15)N by NR is a major determinant of isotopic differences among N pools. Here, we measured in vitro (15)N discrimination by several NRs purified from plants, fungi, and a bacterium to determine the intrinsic (15)N discrimination by the enzyme and to evaluate the validity of measurements made using (15)N-enriched NO(-) 3. Observed NR isotope discrimination ranged from 22 to 32‰ (kinetic isotope effects of 1.022-1.032) among the different isozymes at natural abundance (15)N (0.37%). As the fractional (15)N content of substrate NO(-) 3 increased from natural abundance, the product (15)N fraction deviated significantly from that expected based on substrate enrichment and (15)N discrimination measured at natural abundance. Additionally, isotopic discrimination by denitrifying bacteria used to reduce NO(-) 3 and NO(-) 2 in some protocols became a greater source of error as (15)N enrichment increased. We briefly discuss potential causes of the experimental artifacts with enriched (15)N and recommend against the use of highly enriched (15)N tracers to study N discrimination in plants or soils.