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Materials and Methods Online
We conducted four separate studies in which we measured whole plant
respiration, mass, and nitrogen (N) of a total of 43 species in field, greenhouse, and
growth chamber settings. Physiological methods were similar in all studies and are
presented below. Details of the environmental conditions and plant species and ages were
as follows.
Field/US tree saplings: Naturally-regenerated saplings (6- to 25-years old, n =
119) of four tree species (two needle-leafed gymnosperms and two broadleafed
angiosperms) were sampled in understory shaded (mean 4% of full light, standard error
0.2% of full light) and small gap (mean of 10% light, standard error of 0.2%) conditions
in a mixed conifer-hardwood forest at the Cloquet Forestry Center, Cloquet, Minnesota,
USA1. Air temperatures during the several weeks prior to the beginning of the
measurements were relatively stable and averaged ≈20 °C (climate data, Cloquet Forestry
Center) with average maximum and minimum temperatures approximately 26 and 13 °C
respectively. Respiration measurements were made at 20 °C.
GH/Tree seedlings: The greenhouse study of first-year seedlings of nine species
of cold temperate trees2,3 included shaded (mean of 5% of full sun light) and unshaded
(mean of 25% of full sun light) treatments within a white-washed greenhouse in St. Paul,
MN, USA, which averaged 25/20 °C (day/night) during the study. Respiration
measurements were made for 165 individuals at 23.5 °C, at several different times
(equivalent to different plant ages) throughout the study.
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GC/Tree seedlings: The growth chamber study of first-year seedlings of five tree
species4-6 was conducted under irradiance treatments that equaled 100% of natural total
daily photon flux density4, with contrasting ambient (370 µmol/mol) and elevated
atmospheric CO2 treatments (580 µmol/mol), and growth under three different
temperature regimes, consisting of 18/12, 24/18 and 30/24 °C day/night temperatures
(with a 16-hour photoperiod). Respiration measurements were made at the end of the
dark period several times throughout the study at the respective night temperatures for
each temperature regime for roughly equal numbers of plants from all species and
treatments.
GH/Herbs: The greenhouse study of first-year seedlings of 32 herbaceous species7
was run under unshaded greenhouse conditions with supplemental lighting (similar in
total to ≈50% of full sun light) and included low N (unamended soil) and high N (N
fertilized) treatments. Temperatures varied during the study but averaged (day/night)
≈25/20 °C. Respiration measurements were made at 24 °C.
Due to neighbor and self-shading, foliage of young herbaceous and woody plants
in relatively open natural conditions can experience a wide range of light microhabitats
(e.g., from 25-95% of full light), hence our high light treatments in the greenhouse and
growth chamber studies represent relatively well-lit field conditions for these species.
Our low light plants in the 9-species greenhouse study and the field saplings were grown
under conditions that represent forest understory or small gap conditions. The greenhouse
and growth chamber experiments involved multiple destructive harvests to enable
concurrent determination of respiration rates and plant dry mass as seedlings grew larger
throughout the studies.
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Measurements
In all four studies on each measurement day we hand excavated a number of
individuals from the soil or growth medium prior to respiration measurements.
Immediately after excavation, the entire root system was covered with wet paper towels.
The entire plant was placed in the dark and if needed (i.e. field-grown plants) plants were
temporarily stored at ≈5º to 7 °C in the dark until being prepared for measurement by
being moved to measurement temperature conditions in the dark (in a controlled
environment chamber). Plants were divided into leaves, stems, and roots in the field
sapling and tree seedling greenhouse experiments, and were separated into aboveground
(leaves plus stems) and belowground components for the greenhouse herbaceous plant
study and the growth chamber study of tree species. All the material was kept hydrated
in the dark at measurement temperature for at least 30 minutes before measurement and
typically measured within 3 hours. Tests for effects of respiration protocols were made
to ensure that conditions and timing did not influence the results. For some of the larger
saplings in the field study and the 9-species tree seedling study, tissues were further
partitioned prior to measurement. In the vast majority of cases, all tissues were measured
for each plant. When such partitioning occurred, stems and roots were separated between
coarse (more than 5-mm diameter) and moderately fine tissues (less than 5-mm
diameter).
In all four studies, measurements of dark respiration were made using infra-red
gas analysis of net CO2 efflux in the dark using the same infrared gas analyzers and
cuvettes (LCA-3 and PLC-C, Analytical Development Co. Ltd., Hoddesdon, UK),
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operated in an open configuration1,3,5-7. Columns of magnesium perchlorate removed
water vapour from the analyzer stream. Plants grown under differing CO2 concentrations
in the tree seedling growth chamber study were measured under the CO2 concentrations at
which they were grown5. Rates of net CO2 efflux from entire root systems free of soil,
and of and stems and leaves (either separated or together) were separately measured in
the dark. Gas exchange measurements were taken after the readings were stable for at
least 5-10 minutes.
Specific respiration rates were calculated by dividing the CO2 flux by the dry
mass of the tissue. Calculations of component respiration (e.g., root respiration per plant,
shoot respiration per plant) were made by multiplying the measured dark respiration rate
per unit dry mass by the dry mass of the component. In the rare cases where more than
one measurement was made for a partitioned tissue (such as roots), the total respiration
and the mass were summed for all such samples. Whole plant respiration per plant and
total dry mass per plant were calculated by adding the component respiration and mass
values, respectively.
All the material was dried in a forced air oven (65-70 °C) and mass was
measured. For the studies involving woody plants, total nitrogen concentrations (N) of
dried and ground tissues (mg N g-1 tissue) were measured using the Kjeldahl Digestion
assay (Research Analytical Laboratory, University of Minnesota). For the study of
herbaceous species, percent N was determined on dried, ground tissue samples using a
Carlo Erba NA1500 CHN analyzer (Carlo Erba Instruments, Milan, Italy). With larger
plants, ground and well-mixed tissue was subsampled for N measurement. Total
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aboveground and total plant N contents were calculated by multiplying the N
concentration (mg/g) by the dry mass of the plant (g).
Field/Japan trees: We compiled published aboveground data for a total of 10
individual Chamaecyparis obtusa trees8,9 from two related field studies of 8- and 18-year
old trees, using nighttime respiration data for the three summer months (June to August,
mean ambient air temperature of ≈22 °C) determined using whole-tree chambers.
Aboveground total N for Chamaecyparis was estimated based on published %N values1012
.
To reconcile measurement temperature differences among studies, we adjusted
respiration rates to a common measurement temperature (24 °C) using a published
temperature model13 that uses a temperature-dependent Q10. In scatterplots of respiration
vs. mass, standardizing respiration for measurement temperature shifts the positions (i.e.,
the elevations) of the data upwards for plants measured at lower temperatures. By
definition this does not change the scaling slope of any set of plants measured at a single
temperature. However, standardizing to a common measurement temperature does result
in a potentially different relationship of respiration to mass or nitrogen for a data set that
includes plants originally measured at different temperatures (i.e., the GC/tree seedling
study).
We also conducted a separate analysis to compare scaling relationships of
respiration rates adjusted to growth temperature conditions. Thus, in the case of fieldgrown and greenhouse plants, we also calculated respiration values adjusted to the mean
ambient growth temperature conditions (e.g, average for the proceeding week or two
weeks) prior to the date of respiration measurements. These estimates are relevant given
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evidence of rapid shifts in the elevation of the respiration, nitrogen, and temperature
relationships in response to the mean temperature experienced in the days to weeks prior
to measurements (i.e. temperature acclimation)13-15.
The different respiration data sets (as assessed at measurement temperature,
adjusted to a common measurement temperature, or adjusted to recent growth
temperatures) lead to virtually identical conclusions. All of the scaling relationships
reported in the paper (using respiration adjusted to a common measurement temperature)
are similar in slope to those assessed using respiration data either at measurement
temperatures (i.e., unadjusted) or adjusted to the recent ambient growth temperature.
Hence, the conclusions of these analyses are similar whether using respiration rates made
at disparate temperatures or adjusted to either growth temperatures or a common
temperature.
Supplemental References:
1. J.-L. Machado, Ph.D. Thesis, University of Minnesota (1999).
2. P. B. Reich, Tjoelker, M.G., Walters, M.B., Vanderklein, D. & Buschena, C. Close
association of RGR, leaf and root morphology, seed mass and shade tolerance in
seedlings of nine boreal tree species grown in high and low light. Funct. Ecol. 12, 327338 (1998).
3. P. B. Reich, Tjoelker, M.G., Walters, M.B., Vanderklein, D. & Buschena, C.
Photosynthesis and respiration rates depend on leaf and root morphology and nitrogen
concentration in nine boreal tree species differing in relative growth rate. Funct.Ecol.
12, 395-405 (1998).
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4. M. G. Tjoelker, Oleksyn, J. & Reich P.B. Temperature and ontogeny mediate growth
response to elevated CO2 in seedlings of five boreal tree species New Phytol. 140,
197-210 (1998).
5. M. G. Tjoelker, Oleksyn, J. & Reich P.B. Acclimation of respiration to temperature
and CO2 in seedlings of boreal tree species in relation to plant size and relative growth
rate. Global Change Biol. 5, 679-691 (1999).
6. M. G. Tjoelker, Reich, P.B. & Oleksyn, J. Changes in leaf nitrogen and carbohydrates
underlie temperature and CO2 acclimation of dark respiration in five boreal tree
species. Plant Cell Environ. 22, 767-778 (1999).
7. P.B. Reich et al. Variation in growth rate and ecophysiology among 34 grassland and
savanna species under contrasting N supply: a test of functional differences. New
Phytol. 157, 617-631 (2003).
8. Yokota, T., Ogawa, K. & Hagihara, A. Dependence of the aboveground respiration of
hinoki cypress (Chamaecyparis obtusa) on tree size. Tree Physiol. 14, 467-479 (1994).
9. Yokota, T. & Hagihara, A. Changes in the relationship between tree size and
aboveground respiration in field-grown hinoki cypress (Chamaecyparis obtusa) trees
over three years. Tree Physiol. 18, 37-43 (1998).
10. Kao, W-Y, Lu, C-S& Chang Y-C. Foliar nutrient dynamics of five dominant plant
species in Yuanyang Lake Nature Preserve, Taiwan. Taiwania 49, 49-56 (2004).
11. Zobel, D. B., Roth, L.F. & Hawk, G.M. Ecology, pathology and management of PortOrford-cedar (Chamaecyparis lawsoniana). USDA Forest Service, General Technical
Report PNW-184. Pacific Northwest Forest and Range Experiment Station, Portland,
OR. 161 p. (1985).
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12. Meerts, P. Mineral nutrient concentrations in sapwood and heartwood: a literature
review. Ann. For. Sci. 59, 713–722 (2002).
13. Atkin, O.K. & Tjoelker, M.G. Thermal acclimation and the dynamic response of plant
respiration to temperature. Trends Plant Sci. 8, 343 (2003).
14. Bolstad, P.V., Reich P. & Lee T. Rapid temperature acclimation of leaf respiration rates in
Quercus alba and Quercus rubra. Tree Physiology 23, 969-976 (2003).