12. Assessing Ecosystem-Level Water Relations Through Stable Isotope Ratio Analyses
187
plications caused by nitric oxides fonned upon
combustion when nitrogenous compounds are present. Whole tissue samples are often used for measurements of time-averaged water use efficiency
even though the 813C of various metabolites can
vary considerably. The choice of what fraction to
measure depends greatly on the purpose of the
study, but in general a-cellulose is a good choice
for infonnation on water relations.
Leaf Sampling Considerations
The isotopic composition within a plant varies due
to differences in source water, environmental effects, genetic differences, biochemical composition, phenology, etc., and a sampling plan must take
all these factors into consideration. Sampling
should be done at the same time of the day as diurnal changes in isotopic composition can occur.
Samples should be collected from the same canopy
position, since differences in microenvironment
will affect the extent of evaporative enrichment in
the leaves. Since incident light levels can be quite
variable throughout a canopy, it is important to be
consistent in sampling only sunlit or shaded leaves
from all four cardinal directions. While leaves have
been commonly used in the past, there is now increasing interest in other tissues and plant parts
(Farquhar et al. 1989). The number of plants sampled is dependent on the research question being
addressed, and will be affected by the amount of
environmental and genetic heterogeneity expected.
It is appealing to bulk samples to save time and
money, but there is likely some infonnation on
population-level genetic variation in isotopic composition that then will be lost.
After harvesting, plant samples should be dried
immediately to avoid changes in chemical composition associated with slow drying of living material. If that is not possible, then freezing the tissue
at time of collection (dry ice works well) and drying
later is adequate. Dried material can be stored at
room temperature for extended periods. Dried tissues are ground to pass a 40-mesh screen or in a
mortar and pestle for analysis of the isotopic composition of organic matter. Finely ground material
bums more unifonnly as well as enhances the efficiency of extraction procedures (e.g., cellulose
purification). Since the amount of tissue required
for analysis is so small «3 mg or <0.1 mg with a
cold finger) care must be taken to ensure sample
homogeneity. Often there will be greater variation
in the repeated analysis of the same "bulk sample"
than in repeated analysis of an individual sample
through the mass spectrometer.
Tree Ring Separation
and Cellulose Purification
Most approaches to tree ring separation involve
simply cutting along visibly delineated rings. With
the aid of a dissecting microscope or a microtome,
it is possible to subdivide annual rings into finer
temporal resolution in order to quantify seasonal
variation in 8 values (Loader et al. 1995). Care must
be taken to ensure that false rings and other anomalies are avoided (Stokes and Smiley 1996). Although destructive, the best way to get at tree rings
is by cutting out an entire cross-section of the bole.
Preliminary sampling should be done when using
increment cores to determine how many cores will
be required to obtain enough material for isotopic
analysis from a typical annual ring. Due to potential
variation along the stem, it is best to sample increment cores from all four cardinal directions (Leavitt
and Long 1986).
There has been a tendency for long-tenn,
multiple-year studies to focus sampling only on acellulose from organic matter. To obtain a-cellulose
from a bulk plant sample (either leaf or tree ring),
various modifications of the Wise (1944) method
have been used. The approach used by Leavitt and
Danzer (1993) allows one to batch process many
samples at once. Basically, the preparation of plant
material into a-cellulose involves a delipification
step (using a Soxlet apparatus), followed by repeated boiling (to remove any water-soluble compounds, such as sugars), bleaching with sodium
chlorite and acetic acid (to remove lignins and proteins), and washing of the sample to produce cellulose (a-cellulose and hemicellulose). Since hemicelluloses have exchangeable 0 atoms, it is
important to remove them in a strong sodium hydroxide solution followed by an acetic acid wash.
The resulting a-cellulose is then ready for 8 18 0
analysis. For 8D analysis, the a-cellulose must first
be nitrated in order to remove the exchangeable H
atoms (see below).
187
plications caused by nitric oxides fonned upon
combustion when nitrogenous compounds are present. Whole tissue samples are often used for measurements of time-averaged water use efficiency
even though the 813C of various metabolites can
vary considerably. The choice of what fraction to
measure depends greatly on the purpose of the
study, but in general a-cellulose is a good choice
for infonnation on water relations.
Leaf Sampling Considerations
The isotopic composition within a plant varies due
to differences in source water, environmental effects, genetic differences, biochemical composition, phenology, etc., and a sampling plan must take
all these factors into consideration. Sampling
should be done at the same time of the day as diurnal changes in isotopic composition can occur.
Samples should be collected from the same canopy
position, since differences in microenvironment
will affect the extent of evaporative enrichment in
the leaves. Since incident light levels can be quite
variable throughout a canopy, it is important to be
consistent in sampling only sunlit or shaded leaves
from all four cardinal directions. While leaves have
been commonly used in the past, there is now increasing interest in other tissues and plant parts
(Farquhar et al. 1989). The number of plants sampled is dependent on the research question being
addressed, and will be affected by the amount of
environmental and genetic heterogeneity expected.
It is appealing to bulk samples to save time and
money, but there is likely some infonnation on
population-level genetic variation in isotopic composition that then will be lost.
After harvesting, plant samples should be dried
immediately to avoid changes in chemical composition associated with slow drying of living material. If that is not possible, then freezing the tissue
at time of collection (dry ice works well) and drying
later is adequate. Dried material can be stored at
room temperature for extended periods. Dried tissues are ground to pass a 40-mesh screen or in a
mortar and pestle for analysis of the isotopic composition of organic matter. Finely ground material
bums more unifonnly as well as enhances the efficiency of extraction procedures (e.g., cellulose
purification). Since the amount of tissue required
for analysis is so small «3 mg or <0.1 mg with a
cold finger) care must be taken to ensure sample
homogeneity. Often there will be greater variation
in the repeated analysis of the same "bulk sample"
than in repeated analysis of an individual sample
through the mass spectrometer.
Tree Ring Separation
and Cellulose Purification
Most approaches to tree ring separation involve
simply cutting along visibly delineated rings. With
the aid of a dissecting microscope or a microtome,
it is possible to subdivide annual rings into finer
temporal resolution in order to quantify seasonal
variation in 8 values (Loader et al. 1995). Care must
be taken to ensure that false rings and other anomalies are avoided (Stokes and Smiley 1996). Although destructive, the best way to get at tree rings
is by cutting out an entire cross-section of the bole.
Preliminary sampling should be done when using
increment cores to determine how many cores will
be required to obtain enough material for isotopic
analysis from a typical annual ring. Due to potential
variation along the stem, it is best to sample increment cores from all four cardinal directions (Leavitt
and Long 1986).
There has been a tendency for long-tenn,
multiple-year studies to focus sampling only on acellulose from organic matter. To obtain a-cellulose
from a bulk plant sample (either leaf or tree ring),
various modifications of the Wise (1944) method
have been used. The approach used by Leavitt and
Danzer (1993) allows one to batch process many
samples at once. Basically, the preparation of plant
material into a-cellulose involves a delipification
step (using a Soxlet apparatus), followed by repeated boiling (to remove any water-soluble compounds, such as sugars), bleaching with sodium
chlorite and acetic acid (to remove lignins and proteins), and washing of the sample to produce cellulose (a-cellulose and hemicellulose). Since hemicelluloses have exchangeable 0 atoms, it is
important to remove them in a strong sodium hydroxide solution followed by an acetic acid wash.
The resulting a-cellulose is then ready for 8 18 0
analysis. For 8D analysis, the a-cellulose must first
be nitrated in order to remove the exchangeable H
atoms (see below).