David T. Tingey

Advancing fine root research with minirhizotrons

Minirhizotrons provide a nondestructive, in situ method for directly viewing and studying fine roots. Although many insights into fine roots have been gained using minirhizotrons, a review of the literature indicates a wide variation in how minirhizotrons and minirhizotron data are used. Tube installation is critical, and steps must be taken to insure good soil/tube contact without compacting the soil. Ideally, soil adjacent to minirhizotrons will mimic bulk soil. Tube installation causes some degree of soil disturbance and has the potential to create artifacts in subsequent root data and analysis. We therefore recommend a waiting period between tube installation and image collection of 6-12 months to allow roots to recolonize the space around the tubes and to permit nutrients to return to pre-disturbance levels. To make repeated observations of individual roots for the purposes of quantifying their dynamic properties (e.g. root production, turnover or lifespan), tubes should be secured to prevent movement. The frequency of image collection depends upon the root parameters being measured or calculated and the time and resources available for collecting images and extracting data. However, long sampling intervals of 8 weeks or more can result in large underestimates of root dynamic properties because more fine roots will be born and die unobserved between sampling events. A sampling interval of 2 weeks or less reduces these underestimates to acceptable levels. While short sample intervals are desirable, they can lead to a potential trade-off between the number of minirhizotron tubes used and the number of frames analyzed per tube. Analyzing fewer frames per minirhizotron tube is one way to reduce costs with only minor effects on data variation. The quality of minirhizotron data should be assessed and reported; procedures for quantifying the quality of minirhizotron data are presented here. Root length is a more sensitive metric for dynamic root properties than the root number. To make minirhizotron data from separate experiments more easily comparable, idiosyncratic units should be avoided. Volumetric units compatible with aboveground plant measures make minirhizotron-based estimates of root standing crop, production and turnover more useful. Methods for calculating the volumetric root data are discussed and an example presented. Procedures for estimating fine root lifespan are discussed.

Simple additive effects are rare: a quantitative review of plant biomass and soil process responses to combined manipulations of CO2 and temperature

In recent years, increased awareness of the potential interactions between rising atmospheric CO2 concentrations ([ CO2 ]) and temperature has illustrated the importance of multifactorial ecosystem manipulation experiments for validating Earth System models. To address the urgent need for increased understanding of responses in multifactorial experiments, this article synthesizes how ecosystem productivity and soil processes respond to combined warming and [ CO2 ] manipulation, and compares it with those obtained in single factor [ CO2 ] and temperature manipulation experiments. Across all combined elevated [ CO2 ] and warming experiments, biomass production and soil respiration were typically enhanced. Responses to the combined treatment were more similar to those in the [ CO2 ]-only treatment than to those in the warming-only treatment. In contrast to warming-only experiments, both the combined and the [ CO2 ]-only treatments elicited larger stimulation of fine root biomass than of aboveground biomass, consistently stimulated soil respiration, and decreased foliar nitrogen (N) concentration. Nonetheless, mineral N availability declined less in the combined treatment than in the [ CO2 ]-only treatment, possibly due to the warming-induced acceleration of decomposition, implying that progressive nitrogen limitation (PNL) may not occur as commonly as anticipated from single factor [ CO2 ] treatment studies. Responses of total plant biomass, especially of aboveground biomass, revealed antagonistic interactions between elevated [ CO2 ] and warming, i.e. the response to the combined treatment was usually less-than-additive. This implies that productivity projections might be overestimated when models are parameterized based on single factor responses. Our results highlight the need for more (and especially more long-term) multifactor manipulation experiments. Because single factor CO2 responses often dominated over warming responses in the combined treatments, our results also suggest that projected responses to future global warming in Earth System models should not be parameterized using single factor warming experiments.

Stress ethylene evolution: A measure of ozone effects on plants

Abstract To determine if ethylene evolution by plants was correlated with ozone stress, a range of plant species and cultivars was exposed to varying ozone concentrations. Following exposure, the plants were encapsulated in plastic bags and incubated for up to 22 h. The stress-induced ethylene that accumulated in the bag was monitored and correlated with the effects of ozone on plants. The rate of stress-induced ethylene production (slope parameter B) as a function of ozone concentration was used as a measure of plant sensitivity. Applying this ranking scale, ponderosa pine, eucalyptus and soybean were the most sensitive species; holly, squash and marigold were least sensitive. There was a good correlation between ozone-induced stress ethylene production and foliar injury. However, the coefficient of variability associated with the ethylene determination was substantially less than with the visual injury estimate. The production of stress-induced ethylene generally lasted for less than 48 h following exposure. The measurement of stress ethylene appeared to be a fast, reliable, sensitive and reproducible technique to measure ozone stress on plants.

Hydroxyl free radical mediated formation of 8-hydroxyguanine in isolated DNA☆☆☆

Formation of 8-hydroxyguanine within calf thymus DNA has been studied after exposure to uv-H2O2 as a hydroxyl free radical generating system. Using high-pressure liquid chromatography with electrochemical detection, we measured the amount of 8-hydroxy-2-deoxyguanosine (8-OHdG) in the enzymatically digested DNA. The 8-OHdG content of uv-exposed DNA increased linearly with increasing H2O2 levels up to 0.03%, above which the rate of increase was less than linear. All hydroxyl free radical scavengers studied (mannitol, ethanol, thiourea, and salicylate), if present in the system when DNA was exposed to uv-H2O2, caused a decrease in the amount of 8-OHdG formed. Thiourea when incubated with damaged DNA caused a loss of 8-OHdG when it was an integral part of DNA. In contrast, thiourea did not react with the nucleoside free in solution. Reduced glutathione did not cause a decrease of 8-OHdG, either when it was an integral part of DNA, or, as the free nucleoside in solution.

Effects of environmental conditions on isoprene emission from live oak.

Live-oak plants (Quercus virginiana Mill.) were subjected to various levels of CO2, water stress or photosynthetic photon flux density to test the hypothesis that isoprene biosynthesis occurred only under conditions of restricted CO2 availability. Isoprene emission increases as the ambient CO2 concentration decreased, independent of the amount of time that plants had photosynthesized at ambient CO2 levels. When plants were water-stressed over a 4-d period photosynthesis and leaf conductance decreased 98 and 94%, respectively, while isoprene emissions remained constant. Significant isoprene emissions occurred when plants were saturated with CO2, i.e., below the light compensation level for net photosynthesis (100 μmol m-2 s-1). Isoprene emission rates increased with photosynthetic photon flux density and at 25 and 50 μmol m-2 s-1 were 7 and 18 times greater than emissions in the dark. These data indicate that isoprene is a normal plant metabolite and not — as has been suggested — formed exclusively in response to restricted CO2 or various stresses.

Estimates of Isoprene and Monoterpene Emission Rates in Plants

A range of plant species, including crops, shrubs, herbs, and trees, was surveyed to determine the magnitude of isoprene emissions. In studies to determine if plants emitted isoprene, greenhouse-grown plants were encapsulated in impermeable plastic bags and kept in a growth chamber for 2 h at 30 C and a photosynthetic photon flux density of ca. 350 μE m-2 s-1. To estimate emission rates, greenhouse-grown plants were conditioned in a growth chamber and transferred to a controlled-environment gas-exchange chamber. Gas samples from either the encapsulation bags or gas-exchange chamber were collected, concentrated cryogenically, and analyzed by gas-liquid chromatography. The occurrence of isoprene and monoterpenes was confirmed by combined gas chromatography-mass spectrometry. Of the 54 plant species tested, 37 emitted isoprene. Isoprene emission rates (28 C and 1,000 μE m-2 s-1) for 16 species ranged from 0 to 38.5 μg carbon g-1 h-1. Monoterpenes were detected from six species and emission rates ranged from 0.01 to 3.53 μg carbon g-1 h-1. The encapsulation technique permitted rapid identification of species that emitted isoprene. The emission rate data confirmed the preliminary isoprene ranking and demonstrated the differences between monoterpene and isoprene emissions.

Evaluation of ozone exposure indices in exposure-response modeling

In exposure-response modeling, a major concern is the numerical definition of exposure in relating crop loss to O3, yet few indices have been considered. This paper addresses research in which plant growth was regressed for soybean, wheat, cotton, corn, and sorghum against 613 numerical exposure indices using the Box-Tidwell model. When the minimum sum of squared errors criterion was used, optimum performance was not attained for any single index; however, near optimum performances were achieved by two censored cumulative indices and from a class of indices called the generalized, phenologically weighted, cumulative impact indices (GPWCIs). The top-performing GPWCIs accumulated concentrations, used sigmoid weighting schemes emphasizing O3 concentrations of 0.06 ppm (118 microg m(-3)) or higher, and had phenological weighting schemes with greatest weight occurring 20 to 40 days prior to crop maturity. These findings indicate that (1) peak concentrations are important, but lower concentrations should be included in the calculations, (2) increased plant sensitivity occurs between flowering and maturity, and (3) plants respond to cumulative exposure impact.

A programmable exposure control system for determination of the effects of pollutant exposure regimes on plant growth

Abstract A field-exposure research facility was constructed to provide a controlled environment to determine the influence of the various components of ozone exposure (concentration, frequency and duration) on plant response. The facility uses modified open-top chambers and an automated control system for continuous delivery and control of single or multiple pollutants over a growing season. Numerous exposure profiles (e.g. various episodic regimes, daily peak profiles with sinusoidal-type or square-wave type peaks) can be produced and controlled in all chambers. Ozone is produced by commercially available generators; their outputs are controlled by an HP 41CV hand-held computer through a Hewlett-Packard Interface Loop System (HP-IL). Chamber microenvironmental data and ozone concentration data are collected continuously with a data acquisition system that includes mean hourly ozone concentration, air and soil temperature, relative humidity and solar radiation. The hourly ozone concentration in each chamber ranged from 2 to 12% of the requested concentration over a 174-day season. Initial studies with this facility compared the response of alfalfa and tall fescue growth to episodic and daily peak exposure profiles with equivalent integrated exposure indices over the growing season. Over the period of three cuttings (133 days) alfalfa growth was reduced more when exposed to the episodic profile than with the exposure regime of daily ozone peaks. Tall fescue growth was reduced only slightly over a period of three cuttings (90 days) when exposed to either regime.

Conditions influencing yield and analysis of 8-hydroxy-2′-deoxyguanosine in oxidatively damaged DNA

We have conducted studies to obtain practical knowledge regarding the stability, digestion, and analytical determination of the content of 8-hydroxy-2-deoxy-guanosine (8-OHdG) in oxidatively damaged DNA. Utilizing H2O2 plus uv light to form oxidatively damaged DNA, we found that storage of the DNA at -20 degrees C at alkaline pH caused a significant loss of 8-OHdG, whereas storage at -20 degrees C at neutral or acidic pH prevented loss of 8-OHdG. The 8-OHdG within DNA is stable at 100 degrees C for at least 15 min. Formation of 8-OHdG within DNA using uv light and H2O2 as a hydroxyl free radical-generating system yields the highest amounts when low levels of phosphate buffer are used; but the use of Tris or citrate buffers causes a lower yield of 8-OHdG because these buffers act as scavengers for the hydroxyl free radicals. Independent assessment of hydroxyl free radical flux by the use of salicylate trapping allows assessment of competitive radical reactions. Ethanol washing of plastic microfuge tubes prior to DNA enzymatic digestion improved the yield of 8-OHdG and reduced the variability between samples. Digestion of the oxidatively damaged DNA by the use of a method involving DNase I, endonuclease, phosphodiesterase, and alkaline phosphatase produced the highest yield of 8-OHdG.

Rising atmospheric CO2 and carbon sequestration in forests

Rising CO2 concentrations in the atmosphere could alter Earths climate system, but it is thought that higher concentrations may improve plant growth through a process known as the “fertilization effect”. Forests are an important part of the planets carbon cycle, and sequester a substantial amount of the CO2 released into the atmosphere by human activities. Many people believe that the amount of carbon sequestered by forests will increase as CO2 concentrations rise. However, an increasing body of research suggests that the fertilization effect is limited by nutrients and air pollution, in addition to the well documented limitations posed by temperature and precipitation. This review suggests that existing forests are not likely to increase sequestration as atmospheric CO2 increases. It is imperative, therefore, that we manage forests to maximize carbon retention in above- and belowground biomass and conserve soil carbon.