Harvard Forest Research

Effects of long-term N addition on root physiology

Principal Investigator: David Eissenstat
Pennsylvania State University: May 01 2008 - Dec 01 2010:

Abstract:
Questions: Is N-addition inducing enhanced root oxidative stress? Is it one factor contributing to increased root mortality rate in forests of Northeastern US?
Approach: Use ingrowth cores to collect relatively young roots. Measure root metabolic activity (respiration), membrane integrity (electrolyte leakage), root N concentration (elemental analysis), and the enzymes associated with oxidative stress (CAT, SOD, MDA) from roots preserved in liquid N.
Ingrowth cores: Root ingrowth cores will be made out of plastic tubing mesh (2-3 inch diameter, 15cm deep), buried vertically in the soil after taking soil cores out. Typically, 3 cores will be placed in each experimental plot. Soil will be sieved and placed back in ingrowth container. The ingrowth cores will allow new roots to grow in over a two-month period. During harvest of ingrowth cores, roots around the core will be carefully cut and removed and the ingrowth core will be removed.
Plots:
Bear Brook: 2 N-addition levels (control & 25.2 kg hr-1yr-1), each with 3 replicate plots
Mt. Ascutney: 3 N-addition levels will be used out of 5 (control, 15.7 kg hr-1yr-1 & 31.4 kg hr-1yr-1), each with 2 replicate plots
Harvard Forest: 3 N-addition levels (control, 50 kg hr-1yr-1 & 150 kg hr-1yr-1), each with a replicate megaplots
Number of cores: 3 cores per each replicate plot
Time: Soil coring and ingrowth core installation: 19 May -25 May
Harvesting ingrowth cores: 7 July – 20 July
Analyses:
Field analyses of root tissues from soil cores: Ingrowth core will be removed and separated by horizon if necessary. Roots will be sieved from the soil and carefully washed. Then a pooled sample of roots with the finest three orders will be used for an electrolyte leakage (electrical conductivity) and root respiration measurement (Hansatech oxygen electrode). Electrolyte leakage is a measurement of cell membrane integrity, which relates to root vitality under environmental stress. Root respiration is a measurement of root metabolic activity. Remaining roots will be immediately frozen with liquid N.
Laboratory analyses of root tissues from ingrowth cores: the following measurements will be made on newly grown fine roots frozen in liquid N.
1. Antioxidant enzyme activities: SOD (superoxide dismutases); CAT (catalases). Measurement of enzyme activities will follow the protocol of Massimo (2008), using NAP-25 Column (Amersham Biosciences) for desalting and Pierce BCA Protein Assay Kit (Thermo Scientific).
2. Lipid peroxidation: MDA (malondialdehyde) content
MDA (measured in nmol g-1FW) is the end product of lipid peroxidation, and is recognized as a biomarker of oxidative damage to plant cells. MDA content will be determined following the methods of Draper and Hadley (1990), and Du and Bramlage (1992).
3. N concentration: Elemental analysis
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