Nitrogen fixation by Robinia pseudoacacia (L.) seedlings in the greenhouse under manipulated nitrogen and light treatments

Metadata:
Identification_Information:
Citation:
Citation_Information:
Originator: Ottinger, Sarah L.
Originator: Miniat, Chelcy Ford
Originator: Wurzburger, Nina
Publication_Date: 2023
Title:
Nitrogen fixation by Robinia pseudoacacia (L.) seedlings in the greenhouse under manipulated nitrogen and light treatments
Geospatial_Data_Presentation_Form: tabular digital data
Publication_Information:
Publication_Place: Fort Collins, CO
Publisher: Forest Service Research Data Archive
Online_Linkage: https://doi.org/10.2737/RDS-2023-0007
Description:
Abstract:
Symbiotic nitrogen fixation (SNF) is a critical mechanism of ecosystem recovery, and in forests of the eastern United States, the most common tree species that supports SNF is black locust (Robinia pseudoacacia L.). Despite its prevalence, black locust’s fixation strategy—whether it maintains fixation at a constant rate (obligate fixation) or reduces its fixation rate (facultative fixation)—is unknown. In this study we examined how nitrogen (N) and light control SNF by black locust, by growing seedlings under two nitrogen levels (nitrogen added and nitrogen not added) and across four levels of light (10%, 20%, 40%, and 100%), with 12-13 replicates for each nitrogen x light treatment. This experiment, conducted at the Coweeta Hydrologic Laboratory in North Carolina, started in May 2020 and seedlings were harvested at the end of 12 weeks. This data publication includes the biomass measurements, net photosynthesis, and nodule activity for each replicate from each of 8 light transmittance and nitrogen treatments.
Purpose:
We aimed to examine how light levels and nitrogen addition affected nodulation and nitrogen fixation by Robinia pseudoacacia seedlings.
Supplemental_Information:
For more information about this study and these data, see Ottinger et al. (2023).
Time_Period_of_Content:
Time_Period_Information:
Range_of_Dates/Times:
Beginning_Date: 202005
Ending_Date: 202010
Currentness_Reference:
Ground condition
Status:
Progress: Complete
Maintenance_and_Update_Frequency: None planned
Spatial_Domain:
Description_of_Geographic_Extent:
This experiment took place in North Carolina at the Coweeta Hydrologic Laboratory greenhouse, which is just west of mail conference center building.
Bounding_Coordinates:
West_Bounding_Coordinate: -83.4305652
East_Bounding_Coordinate: -83.4305652
North_Bounding_Coordinate: 35.0596866
South_Bounding_Coordinate: 35.0596866
Bounding_Altitudes:
Altitude_Minimum: 690
Altitude_Maximum: 690
Altitude_Distance_Units: meters
Keywords:
Theme:
Theme_Keyword_Thesaurus: ISO 19115 Topic Category
Theme_Keyword: biota
Theme:
Theme_Keyword_Thesaurus: National Research & Development Taxonomy
Theme_Keyword: Ecology, Ecosystems, & Environment
Theme_Keyword: Plant ecology
Theme_Keyword: Soil
Theme_Keyword: Forest & Plant Health
Theme:
Theme_Keyword_Thesaurus: None
Theme_Keyword: Robinia pseudoacacia (black locust)
Theme_Keyword: succession
Theme_Keyword: disturbance
Theme_Keyword: nodule
Theme_Keyword: rhizobia
Theme_Keyword: ecology
Theme_Keyword: vegetation
Theme_Keyword: early successional
Theme_Keyword: nitrogen fixation
Theme_Keyword: nodules
Theme_Keyword: aboverground biomass
Theme_Keyword: belowground biomass
Theme_Keyword: net photosynthesis
Theme_Keyword: acetylene reduction assay
Theme_Keyword: 15N2 incubations
Place:
Place_Keyword_Thesaurus: None
Place_Keyword: Coweeta Hydrologic Laboratory
Place_Keyword: North Carolina
Place_Keyword: Southern Appalachians
Place_Keyword: Appalachian Mountains
Taxonomy:
Keywords/Taxon:
Taxonomic_Keyword_Thesaurus:
None
Taxonomic_Keywords: single species
Taxonomic_Keywords: plants
Taxonomic_System:
Classification_System/Authority:
Classification_System_Citation:
Citation_Information:
Originator: ITIS
Publication_Date: 2023
Title:
Integrated Taxonomic Information System
Geospatial_Data_Presentation_Form: on-line database
Other_Citation_Details:
Retrieved [January, 11, 2023]; CC0
Online_Linkage: https://www.itis.gov
Online_Linkage: https://doi.org/10.5066/F7KH0KBK
Taxonomic_Procedures:
Taxonomic_Classification:
Taxon_Rank_Name: Kingdom
Taxon_Rank_Value: Plantae
Applicable_Common_Name: plantes
Applicable_Common_Name: Planta
Applicable_Common_Name: Vegetal
Applicable_Common_Name: plants
Taxonomic_Classification:
Taxon_Rank_Name: Subkingdom
Taxon_Rank_Value: Viridiplantae
Applicable_Common_Name: green plants
Taxonomic_Classification:
Taxon_Rank_Name: Infrakingdom
Taxon_Rank_Value: Streptophyta
Applicable_Common_Name: land plants
Taxonomic_Classification:
Taxon_Rank_Name: Superdivision
Taxon_Rank_Value: Embryophyta
Taxonomic_Classification:
Taxon_Rank_Name: Division
Taxon_Rank_Value: Tracheophyta
Applicable_Common_Name: vascular plants
Applicable_Common_Name: tracheophytes
Taxonomic_Classification:
Taxon_Rank_Name: Subdivision
Taxon_Rank_Value: Spermatophytina
Applicable_Common_Name: spermatophytes
Applicable_Common_Name: seed plants
Applicable_Common_Name: phanU+00E9rogames
Taxonomic_Classification:
Taxon_Rank_Name: Class
Taxon_Rank_Value: Magnoliopsida
Taxonomic_Classification:
Taxon_Rank_Name: Superorder
Taxon_Rank_Value: Rosanae
Taxonomic_Classification:
Taxon_Rank_Name: Order
Taxon_Rank_Value: Fabales
Taxonomic_Classification:
Taxon_Rank_Name: Family
Taxon_Rank_Value: Fabaceae
Applicable_Common_Name: peas
Applicable_Common_Name: legumes
Taxonomic_Classification:
Taxon_Rank_Name: Genus
Taxon_Rank_Value: Robinia
Applicable_Common_Name: locust
Taxonomic_Classification:
Taxon_Rank_Name: Species
Taxon_Rank_Value: Robinia pseudoacacia
Applicable_Common_Name: black locust
Applicable_Common_Name: false acacia
Applicable_Common_Name: yellow locust
Access_Constraints: None
Use_Constraints:
These data were collected using funding from the U.S. Government and can be used without additional permissions or fees. If you use these data in a publication, presentation, or other research product please use the following citation:

Ottinger, Sarah L.; Miniat, Chelcy Ford; Wurzburger, Nina. 2023. Nitrogen fixation by Robinia pseudoacacia (L.) seedlings in the greenhouse under manipulated nitrogen and light treatments. Fort Collins, CO: Forest Service Research Data Archive. https://doi.org/10.2737/RDS-2023-0007
Point_of_Contact:
Contact_Information:
Contact_Organization_Primary:
Contact_Organization: Odum School of Ecology, University of Georgia
Contact_Person: Dr. Nina Wurzburger
Contact_Position: Associate professor
Contact_Address:
Address_Type: mailing and physical
City: Athens
State_or_Province: GA
Postal_Code: 30602
Country: USA
Contact_Electronic_Mail_Address: ninawurz@uga.edu
Data_Set_Credit:
This research was supported by the USDA Forest Service, Southern Research Station; USDA Forest Service, Rocky Mountain Research Station; and the National Science Foundation awards DEB-1440485 and DEB-1637522 from the Long-Term Ecological Research (LTER) Program to the Coweeta LTER.


Author Information:

Sarah L. Ottinger
U.S. Department of Energy, UT-Battelle, Oak Ridge National Laboratory
https://orcid.org/0000-0002-3011-8523

Chelcy Ford Miniat
USDA Forest Service, Rocky Mountain Research Station
https://orcid.org/0000-0002-3266-9783

Nina Wurzburger
University of Georgia, Odum School of Ecology
https://orcid.org/0000-0002-6143-0317
Cross_Reference:
Citation_Information:
Originator: Ottinger, Sarah L.
Originator: Miniat, Chelcy F.
Originator: Wurzburger, Nina
Publication_Date: 2023
Title:
Nitrogen and light regulate symbiotic nitrogen fixation by a temperate forest tree
Geospatial_Data_Presentation_Form: journal article
Series_Information:
Series_Name: Oecologia
Issue_Identification: 2023: 1-10
Online_Linkage: https://doi.org/10.1007/s00442-023-05313-0
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Data_Quality_Information:
Attribute_Accuracy:
Attribute_Accuracy_Report:
Error checking was done both visually, and using published literature (e.g., known physiological limits for photosynthesis). The number of decimal places used for each variable convey accuracy of the instrument used.
Logical_Consistency_Report:
The data are logically consistent. The consistency was verified as part of the quality assurance that occurred during data analysis.
Completeness_Report:
A "." was entered in cases where data collection was not possible or relevant, otherwise data are complete. Variable descriptions also provide this information.
Lineage:
Methodology:
Methodology_Type: Field
Methodology_Description:
MATERIAL AND METHODS

We conducted a greenhouse experiment using black locust seedlings at the USDA Forest Service, Coweeta Hydrologic Laboratory in western North Carolina, USA. In May 2020, we germinated Robinia pseudoacacia seeds from an eastern U.S. seed source (Sheffield Seed Company, Locke, NY) and grew them for 12 weeks before the start of the experiment. We grew seedlings in seed trays in a 1:1 ratio by volume of vermiculite and sand inoculated with a native soil slurry from local black locust trees. When seedlings were approximately 3 centimeters (cm) tall, we transplanted them into individual 4-liter (L) pots containing a 1:1 ratio by volume of vermiculite and peat and trace amounts of sand. Soil pH at the end of the experiment was similar between treatments and ranged from 4.8–5.5, which is similar to local field soils.


TIMELINE

April 30, 2020 - Robinia pseudoacacia (L.) (ROPS) seeds were planted in vermiculite/sand seed boxes after scarification by hot water
May 14, 2020 - seedlings germinated and emerged visibly
July 16 and July 24, 2020 - seedlings were delivered to the greenhouse at Coweeta in individual pots
August 4, 2020 (approximately 13 weeks since planting, approximately 12 weeks since germination) - began nitrogen treatments and the experiment started for ROPS
September 28, 2020 - N Hoagland solution applied to trees
October 23, 2020 (11.5 weeks since experiment began) - harvest for ROPS experiment


TREATMENTS

We established eight treatments (two levels of nitrogen (N) crossed by four levels of light, described below), with n = 13, except the -N (no nitrogen addition), 40% light transparency treatment was n = 12. We randomly stratified seedlings into treatments based on initial seedling height and diameter. At the start of treatment, we measured seedling height to the apical meristem and the stem diameter at 5 cm height to estimate total aboveground biomass using data from our previously established allometric equations (Wurzburger and Miniat 2014).

For twelve weeks, we grew seedlings individually in 4-L pots, randomly rearranging their bench position within each treatment every two weeks. During the experiment, average greenhouse air temperature was 22.2 degrees Celsius (ºC) (range 21.1–26.7 ºC). Average soil moisture was 35% v/v (percent volume per volume) (CS655, Campbell Scientific Inc., Logan UT; n = 1 per treatment). Average air relative humidity was 42% (HMP50, Campbell Scientific Inc., Logan, UT; n = 1 per light treatment). Air temperature, soil moisture, and relative humidity did not differ among the treatments.

In addition to a control 100% light transmittance group, we created three additional light levels (40%, 20%, and 10%) using shade cloth. Light levels were above the light compensation point for black locust on an average day in the greenhouse. Because the response of photosynthesis to light is often curvilinear due to the saturating effect of light (Evans et al. 1993), we selected shade cloth that would confer relatively low light levels—levels above the light compensation point for this species and increasing such that we would likely see a linear increase in photosynthesis with our treatment levels. We monitored photosynthetically active photon flux density (PPFD, micromole per second per square meter [µmol m² s⁻¹]; LI-250A with LI-193 sensor, Li-Cor, Lincoln, NE) in each treatment to validate treatment levels (see Harvest sampling: Anet).

Nutrient treatments included two levels of nitrogen as a volumetric application rate, applied as ammonium nitrate in 100 milliliters (mL) every two weeks. The +N treatment consisted of 10 grams of N per year per square meter (g m⁻² y⁻¹), and the -N treatment consisted of no N addition. To ensure that other nutrients were not limiting seedling growth, we added 100 mL of a 50% strength Hoagland’s solution without nitrogen (Hoagland and Arnon 1950) to all pots midway through the experiment.


HARVEST SAMPLING: BIOMASS

After twelve weeks of treatment, we destructively harvested the seedlings to determine nodule biomass, plant growth, and rates of SNF and photosynthesis. Over three consecutive days, we iteratively harvested three replicates from each of the eight treatments until all seedlings were harvested. We separated the aboveground system from the belowground system using shears and washed the plant biomass with tap water. Leaf and woody biomass were separated manually with shears, as were root and nodule biomass with shears and forceps, respectively. Nodules were excised with approximately 0.5 cm of root tissue remaining on either side of the attachment point. Nodules were stored in humidified jars briefly (15 minutes maximum) until activity could be assessed (described below). We dried all biomass at 65 °C for at least 14 days and weighed it to the nearest 0.001 gram (g). We calculated biomass growth over the duration of the experiment by totaling end biomass and subtracting estimated initial biomass.


HARVEST SAMPLING: NITROGENASE ACTIVITY

To determine the nitrogen fixation rate of nodules, we used an acetylene reduction assay (ARA) method paired with 15N2 incubations (Wurzburger et al. 2022). Immediately after harvest, a subset of nodules (mean of 38 milligrams [mg] of dried nodule biomass) was placed into a sealed 250-mL glass jar with a rubber septum, and 10% of the headspace was replaced with acetylene (C₂H₂). We generated acetylene each day of the harvest by reacting calcium carbide with water and stored the gas in evacuated sampling bags. We sampled 10 mL of mixed headspace at the beginning of the incubation and again after 30 minutes, and stored samples in evacuated 20-mL glass vials. Using a gas chromatograph (Model 8610C, SRI Instruments, Torrance, California USA) with a flame-ionization detector, we determined ethylene (C₂H₄) production of each set of nodules by analyzing peaks against known standards. To correct for any potential ethylene contamination, we ran blank samples with acetylene and no nodules as well as nodules with no acetylene; we did not detect ethylene. On seven seedlings, we also performed paired 15N2 incubations on a subset of nodules (Wurzburger et al. 2022). We replaced 10% of the jars’ headspace with 99 atom % 15N2 (Sigma Aldrich, Missouri, USA) and incubated nodules for 30 minutes. All nodules were dried at 60 ºC as above, and those associated with the paired 15N2 incubations were ground to a fine powder and analyzed for δ15N with an isotope ratio mass spectrometer at the University of Georgia Center for Applied Isotope Studies. We used unenriched nodules from each seedling to calculate the C₂H₄:N₂ conversion factor. The biomass of nodules used in acetylene reduction assays and 15N2 incubations were mathematically added to the total nodule biomass. Because not all nodules had detectable nitrogenase activity, we categorized nodule biomass at the plant level into two groups: nodules, which included all seedlings with nodules, regardless of their activity, and active nodules, which only included seedlings that had nodules with detectable activity.


HARVEST SAMPLING: ANET

We conducted leaf gas exchange measurements (LI-6400-40, Li-Cor Biosciences, Lincoln, NE) in tandem with ARA incubations during harvest so that measurements could be paired. We measured net photosynthesis (Anet, micromoles of carbon dioxide per square meter of leaf area per second [µmol CO2 m−2 leaf area s−1]) on the terminal leaflet of the fully extended leaf closest to the apical meristem. Leaf temperature was set to 23 °C, CO2 in the sample cell was 435 micromole per mole (µmol/mol), flow rate was 400 micromoles per second (µmol s−1), and relative humidity was between 68–75%. Light in the sample chamber was set to ambient PPFD prior to measurement and stayed constant during measurement. Average (standard error) PPFD levels in the sample chamber for the light treatments were 47.9 (5.9), 70.4 (5.8), 75.8 (8.9), and 284.3 (19.8) millimole per second per square meter (mmol m⁻² s⁻¹) for the 10%, 20%, 40%, and 100% transmittance treatments. It is notable that the weather during harvest days was overcast, and we supplemented light in the greenhouse with the high-pressure sodium lamps.


For additional information see Ottinger et al. (2023).
Methodology_Citation:
Citation_Information:
Originator: Evans, John R.
Originator: Jakobsen, Iver
Originator: Ögren, Erling
Publication_Date: 1993
Title:
Photosynthetic light-response curves: 2. Gradients of light absorption and photosynthetic capacity
Geospatial_Data_Presentation_Form: journal article
Series_Information:
Series_Name: Planta
Issue_Identification: 189: 191-200
Online_Linkage: https://doi.org/10.1007/BF00195076
Methodology_Citation:
Citation_Information:
Originator: Hoagland, Dennis R.
Originator: Arnon, Daniel I.
Publication_Date: 1950
Title:
The water-culture method for growing plants without soil
Geospatial_Data_Presentation_Form: circular
Series_Information:
Series_Name: California Agricultural Experiment Station
Issue_Identification: Circular 347
Publication_Information:
Publication_Place: Berkeley, CA
Publisher: University of California
Methodology_Citation:
Citation_Information:
Originator: Ottinger, Sarah L.
Originator: Miniat, Chelcy F.
Originator: Wurzburger, Nina
Publication_Date: 2023
Title:
Nitrogen and light regulate symbiotic nitrogen fixation by a temperate forest tree
Geospatial_Data_Presentation_Form: journal article
Series_Information:
Series_Name: Oecologia
Issue_Identification: 2023: 1-10
Online_Linkage: https://doi.org/10.1007/s00442-023-05313-0
Methodology_Citation:
Citation_Information:
Originator: Wurzburger, Nina
Originator: Miniat, Chelcy F.
Publication_Date: 2014
Title:
Drought enhances symbiotic dinitrogen fixation and competitive ability of a temperate forest tree
Geospatial_Data_Presentation_Form: journal article
Series_Information:
Series_Name: Oecologia
Issue_Identification: 174: 1117-1126
Online_Linkage: https://doi.org/10.1007/s00442-013-2851-0
Online_Linkage: https://www.fs.usda.gov/research/treesearch/45385
Methodology_Citation:
Citation_Information:
Originator: Wurzburber, Nina
Originator: Motes, Jessie I.
Originator: Miniat, Chelcy F.
Publication_Date: 2022
Title:
A framework for scaling symbiotic nitrogen fixation using the most widespread nitrogen fixer in eastern deciduous forests of the United States
Geospatial_Data_Presentation_Form: journal article
Series_Information:
Series_Name: Journal of Ecology
Issue_Identification: 110(3): 569-581
Online_Linkage: https://doi.org/10.1111/1365-2745.13819
Online_Linkage: https://www.fs.usda.gov/research/treesearch/63561
Process_Step:
Process_Description:
see methodology section
Process_Date: 2022
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Entity_and_Attribute_Information:
Overview_Description:
Entity_and_Attribute_Overview:
Below you will find a list and description of the files included in this data publication.

VARIABLE DESCRIPTION FILE (1)

1. \Data\_variable_descriptions.csv: Comma-separated values (CSV) file containing a list and description of variables found in all data files. (A description of these variables is also provided below.)

Columns include:

Filename = name of data file

Variable = name of variable

Units = units (if applicable)

Precision = precision (if applicable)

Description = description of variable



DATA FILES (1)

1. \Data\ROPS_GH_data_Ottinger.csv: CSV file containing experimental biomass, net photosynthesis, and nodule activity data from Robinia pseudoacacia seedlings grown under 8 treatment conditions representing 2 Nitrogen addition conditions and 4 light transmittance conditions.

Variables include:

Sample name = Sample name

Replicate = Number of replicate within the sample

Light transmittance = Percent light transmittance (percent [%]) (Note: original PPFD was measured to 0.1 mmol photons m-2 area s-1)

Nitrogen treatment = Nitrogen addition treatment (1 = nitrogen [N] was added, 0 = no N added)

Total nodule biomass = Total nodule biomass (grams [g], precision = 0.001)

Aboveground biomass = Aboveground biomass (g, precision = 0.001)

Belowground biomass = Belowground biomass (g, precision = 0.001)

Stem biomass = Stem biomass (g, precision = 0.001)

Leaf biomass = Leaf biomass (g, precision = 0.001)

Total plant biomass = Total plant biomass (g, precision = 0.001)

Net photosynthesis = Net photosynthesis ("." = no suitable leaves to measure) (micromoles of carbon dioxide/square meter leaf area/second [µmol CO₂ m⁻² leaf area s-1], precision = 0.0001)

Fixation = Rate of fixation ("." = no nodules, 0 = nodules were not active) (micromoles of Ethlyene fixed/grams nodule/hour [µmol C₂H₄ fixed/g nodule/hour])
Nodule presence = Presence of nodules (1 = present, 0 = absent)

Total biomass growth = Total biomass growth calculated as the accumulation over the duration of the experiment (end biomass subtracted from estimated initial biomass) (g, precision = 0.001)

Aboveground growth = Aboveground growth calculated as the accumulation over the duration of the experiment (end biomass subtracted from estimated initial biomass) (g, precision = 0.001)

Belowground growth = Belowground growth calculated as the accumulation over the duration of the experiment (end biomass subtracted from estimated initial biomass) (g, precision = 0.001)
Entity_and_Attribute_Detail_Citation:
Ottinger, Sarah L.; Miniat, Chelcy F.; Wurzburger, Nina. 2023. Nitrogen and light regulate symbiotic nitrogen fixation by a temperate forest tree. Oecologia. 2023: 1-10. https://doi.org/10.1007/s00442-023-05313-0
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Distribution_Information:
Distributor:
Contact_Information:
Contact_Organization_Primary:
Contact_Organization: USDA Forest Service, Research and Development
Contact_Position: Research Data Archivist
Contact_Address:
Address_Type: mailing and physical
Address: 240 West Prospect Road
City: Fort Collins
State_or_Province: CO
Postal_Code: 80526
Country: USA
Contact_Voice_Telephone: see Contact Instructions
Contact Instructions: This contact information was current as of February 2023. For current information see Contact Us page on: https://doi.org/10.2737/RDS.
Resource_Description: RDS-2023-0007
Distribution_Liability:
Metadata documents have been reviewed for accuracy and completeness. Unless otherwise stated, all data and related materials are considered to satisfy the quality standards relative to the purpose for which the data were collected. However, neither the author, the Archive, nor any part of the federal government can assure the reliability or suitability of these data for a particular purpose. The act of distribution shall not constitute any such warranty, and no responsibility is assumed for a user's application of these data or related materials.

The metadata, data, or related materials may be updated without notification. If a user believes errors are present in the metadata, data or related materials, please use the information in (1) Identification Information: Point of Contact, (2) Metadata Reference: Metadata Contact, or (3) Distribution Information: Distributor to notify the author or the Archive of the issues.
Standard_Order_Process:
Digital_Form:
Digital_Transfer_Information:
Format_Name: CSV
Format_Version_Number: see Format Specification
Format_Specification:
Comma-separated values file
File_Decompression_Technique: Files zipped with 7-Zip 19.0
Digital_Transfer_Option:
Online_Option:
Computer_Contact_Information:
Network_Address:
Network_Resource_Name: https://doi.org/10.2737/RDS-203-0007
Fees: None
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Metadata_Reference_Information:
Metadata_Date: 20230213
Metadata_Contact:
Contact_Information:
Contact_Organization_Primary:
Contact_Organization: Odum School of Ecology, University of Georgia
Contact_Person: Dr. Nina Wurzburger
Contact_Position: Associate professor
Contact_Address:
Address_Type: mailing and physical
City: Athens
State_or_Province: GA
Postal_Code: 30602
Country: USA
Contact_Electronic_Mail_Address: ninawurz@uga.edu
Metadata_Standard_Name: FGDC Biological Data Profile of the Content Standard for Digital Geospatial Metadata
Metadata_Standard_Version: FGDC-STD-001.1-1999
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