Current Research

Fate of Nitrogen in Ferrum's Watershed

Measurements of nitrogen in plant, soil, soil water, and the creek are being correlated with pasture fertilization and weather data. Ultimately, the data will be compared to data collected at several watersheds in the appalachian region as part of ongoing research at collaborating colleges.

Evaluation of Switchgrass Cultivars for Persistence and Biomass Potential

The use of field crops as an energy source has received great attention in recent years for two main reasons: (1) the need to reduce our dependency on fossil fuels as an energy source and (2) our need to mitigate anthropogenic atmospheric carbon release. Switchgrass (Panicum virgatum L.), a warmseason C4 grass, has emerged as a crop with great potential as a biomass production system to generate biofuel if appropriate bioconversion technologies become feasible. As a perennial cropping system, switchgrass is an attractive choice since it produces well on marginal crop land with minimal management. Used as a biofuel, switchgrass would incorporate as much carbon as biomass via photosynthesis from the atmosphere as it would add by burning as a biofuel.

Switchgrass production for biofuel may also replace production systems that have become economically and environmentally difficult to maintain. In southwest Virginia, for example, dairy and beef producers have come under increased scrutiny as nonsource polluters. In addition, the number of dairy producers has dropped by approximately 50% since 1990. Tobacco producers are another farming sector particularly hard hit with a large loss in production due to the reduction in quotas. Switchgrass grown as a bioenergy crop may provide a successful option for producers who must now look for an alternative high value crop with reduced environmental costs.

To be an economically suitable crop for adoption by farmers, two important criteria must be evaluated – production potential with minimal input and plant persistence. The objective of this study, therefore, was to compare biomass and persistence among cultivars of switchgrass grown in infertile soil.

The Effects of Chilling on Maize Photosynthesis after Interrupting the C4 Cycle with an Inhibitor of Phosphoenolpyruvate Carboxylase

Project Summary
Two key photosynthetic enzymes, phosphoenolpyruvate (PEP) carboxylase and ribulose bisphosphate carboxylase/oxygenase (rubisco) were investigated for their role in the reduction of photosynthesis observed in C4 plants following chilling in the light. The C4 cycle was interrupted using a chemical inhibitor in an attempt to isolate the Calvin-Benson cycle during the chilling process.

Rationale
C4 plants occur rarely in cooler climates in which average temperatures during the growing season are less than about 16°C, presumably because photosynthesis is inhibited by low temperatures (Sage et al., 1999). The mechanism for this inhibition is unclear, however, a breakdown of photosynthesis may result because cool temperatures impair the ability of C4 plants to concentrate CO2 via the C4 cycle into bundle sheath cells where the Calvin-Benson cycle operates. Kubien et al. (2003) proposed that C4 plants in cool climates, compared to their C3 competitors, are constrained because they have less rubisco, the enzyme that binds CO2 in the Calvin-Benson cycle. Thus, if the C4 cycle is impaired by cool temperatures, rubisco would not operate at maximum efficiency. In addition, because bundle sheath cells resist CO2 diffusion (Brown and Byrd, 1993), CO2 within bundle sheath cells likely would be very low if the C4 cycle becomes impaired by chilling.

Another possible explanation for inhibition of photosynthesis by cool temperatures is direct impairment of C3 photosynthesis in chilling sensitive species. Following chilling in the light, Calvin-Benson cycle enzymes including rubisco are reduced in tomato and other species of tropical origin (Powles et al., 1983; Byrd et al., 1995). Specifically, rubisco activation in these species is impaired after short term exposure to low temperatures (4°C) and high light, which also includes an inability to regenerate ribulose bisphosphate.

Objective
The objective for this project was to determine how short-term exposure to chilling temperatures in the light affects the activities of the photosynthetic carboxylating enzymes within leaves of the C4 plant maize (Zea mays L.). We attempted to separate the C4 and Calvin-Benson cycles in the leaf by chemically inhibiting PEP carboxylase, the enzyme that initially binds CO2 from the atmosphere in C4 plants, and then measuring PEP carboxylase and rubisco activities. Photosynthetic efficiency was monitored using chlorophyll fluorescence techniques throughout the chilling treatments and in the absence of an operational C4 cycle (that is, in the presence of the PEP carboxylase inhibitor).

Procedure
PEP carboxylase inhibition
Eight young, fully expanded leaves of maize were detached and their bases quickly recut underwater. The leaf bases were placed in small vials of distilled water and exposed to 1000 micromol photons/ m2 s1 for an equilibration period of 30 minutes. For some leaves, distilled water was replaced by 4 mM 3,3-dihydroxyphosphinoylmethylo-2-propenate (DCDP) and allowed to take up the chemical inhibitor for 30 minutes. Both inhibited and uninhibited leaves were then subjected to either 25°C or 4°C. Measurements of chlorophyll fluorescence was determined for each time period. The treatments examined the effects of chilling in the light on leaves with and without the chemical inhibitor of PEP carboxylase as well as subsequent recovery of leaves from the chilling treatment and from DCDP for 18-hr following the 8-hour exposure to chilling in the light and the inhibitor.

Chlorophyll fluorescence
Measurements of chlorophyll fluorescence was determined for each time period with a chlorophyll fluorimeter. Leaves were dark-adapted for 15 minutes prior to each measurement. After dark adaptation, a saturating flash (~8000 micromol/ m2 s1 for 1 s) was applied raising the ground state value (Fo) to its maximum value, Fm. In this condition, the first electron acceptor of PSII is fully reduced, which allowed us to determine the maximum efficiency of PSII using the following equation:

Fv/Fm = (Fm – Fo)/Fm

The maximum PSII efficiency (Fv/Fm) can be used as an indicator of stressful conditions in leaves and was used to measure photosynthetic differences between leaves at 25°C or 4°C and between leaves uninhibited or inhibited by DCDP. After the 8-hour treatments, all segments were placed in low light at 25°C to investigate the recovery from the DCDP treatment and the treatment of low temperature and high light.

Chilling protocol
After the 30-min equilibration with either 4 mM DCDP or distilled water, leaves were cut into 3 cm long segments and placed in Petri plates (tops removed). The Petri plate bottoms contained either distilled water or 4 mM DCDP and were placed into Styrofoam coolers and allowed to float on ice water (4°C treatment) or water kept at room temperature. Above each cooler, the light source was directed onto the leaves with a light intensity of 1000 micromol/l m2 s1. A large glass reservoir positioned between the lamp and leaves served as an infrared filter. Ice was added as needed to replenish the ice water in the cooler . After the 8-hr temperature treatment at high light, ice was removed from the one cooler and replaced with water at room temperature. The light source was adjusted to 100 micromol/ m2 s1 using a rheostat. In addition, leaves treated with DCDP were placed in Petri plates containing water. Temperature were monitored throughout the experiment using thermocouples (Type T) positioned on the underside of randomly selected leaves.

Assays for enzyme activity
From each leaf, a cork borer was used to remove two leaf discs (approximately 2 square centimeters each). Leaf disc samples were frozen in liquid N2, and placed in a -20°C freezer for later assays of rubisco and PEP carboxylase activities. Following the initial samples taken at times -0.5 and 0 hr, leaf discs for rubisco and PEP carboxylase activities were sampled at 4, 8, and 24 after the beginning of the experiment.

For extraction of rubisco, leaf discs were homogenized in a chilled mortar with 30 mg polyvinyl polypyrrolydone, 0.2 g sand, and 4 mL extraction buffer containing 100 mM Bicine (pH 8.2), 1 mM EDTA (ethylenediamine tetra acetic acid) – NaOH (pH 7.0, 5 mM MgCl2, 5 mM DTT (dithiothreitol) and 0.2% bovine serum albumin (BSA). A 50 microliter subsample will be analyzed to determine chlorophyll and protein concentrations. The homogenate was filtered through one layer of Mira cloth and centrifuged for 2 min in a refrigerated microcentrifuge at the highest speed. The extract was kept on ice at all times and immediately assayed. The activity of rubisco was assayed by the appearance of the products (3-phosphoglyceric acid). In this procedure we assayed the ability of maize leaf extracts to produce 3-phosphoglyceric acid by converting this product to glyceraldehyde-3-phosphate using the enzymes phosphoglycerate phosphokinase and glyceraldehyde phosphate dehydrogenase. Phosphoglycerate phosphokinase transfers a phosphate group from ATP to 3-phoshoglyceric acid to form 1,3-bisphosphoglyceric acid. Glyceraldehyde phosphate dehydrogenase reduces 1,3-bisphosphoglyceric acid, oxidizing NADH. The oxidation of NADH was followed spectrophotometrically at 340 nm. For every mole of ribulose-1,5-bisphosphate carboxylated, two moles of NADH were oxidized.

For extraction of PEP carboxylase, leaf discs were homogenized in a chilled mortar and pestle and an extraction medium containing 100 mM TRIS-HCl, pH 7.3, 5 mM MgCl2, 2 mM KH2PO4, 1 mM EDTA, 20% (v/v) glycerol, 10 mM ß-mercaptoethanol, 10 mM NaF, 2 mM PMSF, 5 mM DTT, and 2% (w/v) insoluble PVP. The homogenate was centrifuged at 15,000 g for 5 min. The supernatant was used as crude extract. The activity of PEP carboxylase in the extract was assayed by coupling the reaction to NAD malic dehydrogenase and monitoring NADH oxidation at 340 nm. The assay was performed at 30 °C irrespective of treatment temperature.

Significance of research to the undergraduate
The research provided for the chemistry major who worked on this project a hands-on experience of experimental design and preparation for graduate research in biochemistry. It provided the experience needed to perform enzyme assays and led to a greater understanding of the biochemistry of photosynthesis.

References
Byrd GT, Ort DR, Ogren WL (1995) The effects of chilling in the light on ribulose-1,5-bisphosphate carboxylase/oxygenase activation in tomato. (Lycoperison esculentum Mill.) Plant Physiology 107: 585-591

Brown RH, Byrd GT (1993) Estimation of bundle sheath cell conductance in C4 species and O2 insensitivity of photosynthesis. Plant Physiology 103: 1183-1188

Kubien DS, von Caemmerer S, Furbank RT, Sage RF (2003) C4 photosynthesis at low temperature. A study using transgenic plants with reduced amounts of rubisco. Plant Physiology 132: 1577-1585

Powles SB, Berry JA, Bjorkman O (1983) Interaction between light and chilling temperature on the inhibition of photosynthesis chilling-sensitive plants. Plant Cell and Environment 6: 117-123

Sage RF, Wedin DA, Li M (1999) The biogeography of C4 photosynthesis, patterns and controlling factors. In RF Sage, RK Monson, eds, C4 Plant Biology. Academic Press, Toronto, pp 313–373

Critical nitrogen concentrations in Grass-Legume Mixtures

As crops age, the rate of nitrogen acquisition per unit of ground area declines probably as a result of lowering crop demand for soil nitrogen caused by the internal cycling of nitrogen from aging to young developing tissues and development of more structural tissues. The relationship between the critical nitrogen concentration (the minimum nitrogen concentration required for maximum plant growth rate) and dry matter per unit ground area appears to be similar for a wide range of crops. Greenwood et al. (1990) defined a nitrogen dilution curve for both C3 and C4 crops linking critical nitrogen concentrations in the above ground biomass (N%) and crop dry weight (W) as

N% = aW-b, (W > 1 t/ ha).

For both C3 and C4 crops the model accounted for 86% of the variance when b = 0.5, a =5.7 (C3) and a= 4.1 (C4). Two immediately applicable generalizations result from the model: the relative growth rate is twice the relative nitrogen accumulation rate and crops acquire half of their total nitrogen requirements as they reach 25% of the final weight.

The model has important implications for forages, although growth of forage crops represents a more complicated situation. For example, above ground biomass may be kept low by grazing herbivores and swards are often maintained not as a monoculture but as a botanical mixture. My objective is to determine how best to modify the model when biomass per unit area is low. Because forage quality is best when above ground biomass is kept low , it is important to understand how the model holds up as above ground biomass fluctuates around 1 t/ha. Since my interest concerns regrowth of mowed or grazed swards, I am using the model to determine the time course of nitrogen accumulation in above ground biomass in Tall fescue-white clover and orchardgrass-white clover mixtures. A better understanding of relationships among nitrogen accumulation rates, mixture persistence, forage quality, and critical nitrogen concentrations of botanical mixtures will help better define fertilizer strategies and cutting frequencies.