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BIOLOGICAL MARKERS: NEW AND EMERGING TECHNOLOGIES
Peter J. H. Sharpe
Biosystems Research Group
Department of Industrial Engineering
Texas A & M University
College Station, TX 77843
Widespread, chronic, low concentrations of atmospheric
pollutants lead to circumstances where visible impact upon forests
is generally not evident. For this reason an array of reliable
markers is needed to assess physiological damage to vegetation
long before it becomes visible. Several new and emerging
technologies offer promising techniques for use as markers.
Short-lived radioisotopes (SLR) of metabolizable compounds can
now be introduced to plants and their assimilation, translocation
and allocation patterns characterized in viva, nondestructively, and
in real time as functions of pollutant impact. Nuclear magnetic
resonance (NMR) has recently developed into a powerful tool for
quantitative analysis of metabolic processes in living tissues,
identifying chemical groups and their concentrations. Infrared
reflectance (JR) techniques have been used extensively in
analytical laboratories and are now a viable technique for
physiological measurements in intact plants. Finally, the
development of fiber optics, semiconductor and membrane
technologies has set the stage for the near-future development of
a variety of microsensors capable of making physiological and
biochemical measurements localized to the level of single cells or
tissues. These latter techniques provide even more opportunities
for markers yet to be identified. The implementation of
short-lived radioisotopes to measure biological markers in field
environments is discussed, together with the cost-effectiveness of
emerging technologies.
ABSTRACT
Richard D. Spence
Biosystems Research Group
Department of Industrial Engineering
Texas A & M University
College Station, TX 77843
INTRODUCTION
Air pollutant effects on plants, especially impacts at low concentrations where
visible injury is not evident, have been difficult to assess. Measurements of biochemical
process rates, such as photosynthesis, are confounded by environmental factors as well as
the cryptic nature of plant physiology. The effects of pollutants are offset to some
degree by compensatory mechanisms employed by the plant to overcome limitations in
individual resources (Bloom et al., 1985~. If air pollutants interfere with the acquisition
and/or assimilation of one or more resources, and the plant as a consequence initiates
compensatory shifts in metabolism, cause-and-effect relationships become difficult to
establish.
Compensatory mechanisms, therefore, can hide many direct effects of air pollutants
until the stresses exceed the compensatory limits. At present, there is no method for
determining how near a plant community or even an individual plant is to its
compensatory limit. In addition, the compensatory limit is much more than just a
81
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function of air pollution exposure. It is influenced by the availability of resources for
growth, climatic variables and the structure of biotic interaction within the ecosystem or
controlled experiment. The correlation of damage to dose is appropriate only at higher
levels of exposure above the injury threshold. Below this threshold, the relationship
between dose and response is not evident to an external observer.
NEED FOR BIOLOGICAL MARKERS
If compensatory mechanisms hide the response of plants to low doses of air
pollutants, then the traditional statistically based experimental design approaches are
ineffective. Alternatively, a method of measurement is required to identify and quantify
the degree to which compensatory changes are occurring within the plant. Although
these homeostatic shifts may not result in a reduction in overall yield, they serve as an
estimate of internal stress imposed upon the plant by air pollutants. Further
investigation of internal stress responses can lead to an understanding of how close the
compensatory shifts are to the breakdown point, beyond which further stress leads to
yield reduction or physiological disruption of normal metabolism.
A marker can therefore be defined as an indicator of the physiological strain
experienced by a plant subjected to a range of interacting stresses. A marker may not
be indicative of any external change in plant growth or yield, especially where
anthropogenic stresses mimic the action of natural stresses. Where this occurs, the plant
has an built-in compensatory response partially to overcome limitations imposed in this
manner. For example, if ozone interferes with carbon dioxide- assimilation, the plant
might respond by treating ozone stress as a reduction in CO2 concentration and allocating
a greater proportion of photosynthate to synthesizing Calvin cycle enzymes and new
leaves, until the perceived CO2 limitation was compensated for and equilibrated with
other limitations for water, nutrients and light. If this is accomplished easily, then no
reduction in total yield becomes obvious. If the physiological strain cannot be
compensated then reductions in yield will become evident. Even where yield reductions
can be compensated, the compensation response may leave the plant vulnerable to
drought, insect, disease or other natural catastrophes (Sharpe and Wu, 1985; Sharpe et
al., 1985; Sharpe and Scheld, 1986~.
An approach using markers contrasts with the traditional dose-response approach
because it emphasizes the need for recognizing how basic processess affect plant
performance. The physiological and morphological systems of the plant must be
monitored to reveal how, and to what extent, the plant is affected by air pollution
stresses even when no yield response is evident. The physiological and morphological
measures chosen for monitoring internal changes, then, must be interpreted by
physiologically based metabolic models.
DESIRED CHARACTERISTICS OF MARKERS
Ideally, a biological marker should be easily measured with a minimum of equipment
and skill. In both medical and plant physiology applications, however, the greatest
breakthroughs in monitoring the internal status of living systems have been accomplished
using rather sophisticated biotechnology. In other words, the important markers in
medicine and biology require a considerable investment in technology and expertise. The
second desirable characteristic of a marker is that its interpretation should be
unambiguous. There should be no uncertainty in its level of significance. It should be
obvious when it occurs. Third, a marker should be manifested in such a way that it can
be explained in physiological terms. To have confidence in its predictive capability as an
indicator of stress compensation, a marker should form a component of the scientific
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framework of concepts and knowledge relating to internal biochemical and biophysical
processes. Fourth, physiological markers should foreshadow morphological changes.
Fifth, a marker should allow measurements that are non-destructive, in viva and
observable in real time. With the exception of the high cost of the monitoring
technology, all of the above desirable characteristics for applying markers can be met in
air pollution research using a range of new and emerging technologies.
NEW AND EMERGING TECHNOLOGIES
Although many of the new and emerging technologies have become associated with
medicine, these techniques arose in the biological and bioengineering sciences. Their use
has been most obvious in the medical sciences largely because of the larger funding base
and the need for innovative methods for studying internal processes leading to cancer,
heart disease and other intractable medical problems. There has been no reason why the
plant sciences could not have maintained a lead in many of these ~technologies, especially
for diagnosing the internal effects of air stresses on plants.
The power of personal computers attached to measurement instruments is providing
new techniques to analyze and display quantitative data. Probably the most
sophisticated general application of computers in emerging technologies involves Fourier
transform (FT) spectroscopy. Each application of FT techniques has led to the
development of whole new fields of instruments (Hirschfeld, 1985~. Three examples of
FT-based quantitative analysis are gas chromatography-mass spectroscopy, FT-infrared
(Fl-IR) and FT nuclear magnetic resonance (FT-NMR).
The power of the current approaches to "intelligent" instrumentation is
demonstrated by near infrared reflectance analysis (FT-NIRA). This technique can now
be used for biological analysis (Hirschfeld and Stark, 1984~. The spectral signatures of
various constituents are inaccurately known and heavily overlapped. Instead of
conventional data reduction, NIRA uses a combination of spectral correlation and a
~self-learning" algorithm. This operates by measuring a set of preanalyzed samples,
cross-correlating the spectra to composition by multilinear regression, and using an
optimization algorithm to select a set of measurement wavelengths and calibration
coefficients for the analysis. NIRA is verified with a second, independent, preanalyzed
sample set. This approach provides approximately 0.1 percent repeatable analysis using
data in which a 10 percent compositional variation is barely perceptible to the eye
(Hirschfeld, 1985~.
In addition to IR spectral methods, Sharpe and Scheld (1986) identified three other
emerging technologies for in vivo studies of air pollution effects on plants. These are
(1 ~ short-lived, high-energy radioisotopes (SLR) of metabolizable compounds, (2) NMR and
(3) microsensors using combinations of fiber optics, semiconductors and membrane
techniques. This list is not complete, as new techniques are being developed and adapted
continuously. We focus in this paper on the complementary technologies offered by SLR
and NMR methods.
SHORT-LIVED RADIOISOTOPE TECHNOLOGIES
The role played by isotopes, stable and radioactive, in plant physiology studies is
well established. Long-lived radioisotopes (e.g., 14C and 35S) have been used for some
years (Fares et al., 1983). The use of SLR is comparatively more recent, but the
development of small accelerators, e.g., 3-5 MeV Van de Graaff generators and 14-35
MeV cyclotrons (Fares et al., 1978; McKinney et al., 1988), and the advent of
minicomputers and microprocessors (Fares et al., 1978) for data acquisition and analysis
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have made the production and use of SLR in dynamic plant studies a viable technique.
SLR have many advantages for use as physiological markers: (1) Many SLR are positron
(13+) emitters, e.g., 1 1C, 3N, 150, 30P, which emit two S-rays at 180° to each other with
sufficient energy (0.511 MeV) to penetrate several centimeters of tissue, allowing for in
vivo coincident counting highly localized within the tissue. (2) The short half life of the
isotope makes it possible to conduct several experiments simultaneously or in rapid
succession on the same plant under the same or contrasting sets of environmental
conditions. (3) The half life of the isotope is often comparable to the turnover times of
metabolic pools. (4) The nondestructive nature of the isotope means that the plant can
be used as its own control. (5) The potentially high sensitivities of the experiments
often reveal unsuspected phenomena. Thus, the use of SLR allows for the highly
localized characterization of plant physiological processes which is non-invasive,
nondestructive, in viva and in real time.
In the last decade 1 1 C (half life = 20.4 minutes), in particular, has been effective in
characterizing many plant physiological processes and responses to environmental stresses.
Phloem loading, unloading, concentration and transport speeds have been characterized to
elucidate controlling mechanisms (Minchin and Thorpe, 1982; Goeschl et al., 1984,
Magnuson et al., 1986~. Phloem transport has been shown to be severely affected by
temperature, especially cold temperature (Potvin et al., 1984; 1985; Goeschl et al., 1984)
and by plant exposure to ozone (Sharpe et al., 1988). These last studies have all
confirmed that 1 C makes an excellent marker in studies of plant response to stress
because it documents the severity of physiological strains long before visible damage
occurs.
NUCLEAR MAGNETIC RESONANCE TECHNOLOGIES
NMR began as a technique of analytical chemistry but evolved into a methodology
that has been widely used in molecular biology and studies of biosynthetics of natural
products (Scott, 1985; Bax and Lerner, 1986~. Roberts (1984) reviewed over 20 NMR
studies of in vivo plant metabolism. NMR is one of the most powerful methods for
quantitative analysis of metabolic processes in living tissues. Over the past 10 years,
the sensitivity of commercially available NMR spectrometers has increased dramatically.
The technique is based on placing the living sample in a strong, homogeneous magnetic
field which aligns the nuclear magnets of certain chemical isotopes, such as 1 H. 1 3C, 1 SN
and 3 ~ P. These elements possess a permanent magnetic moment from their nuclear spin.
Irradiation of a live plant with a pulse of the appropriate frequency range excites the
nuclear magnets aligned against the external magnetic field, providing the following
physiological information: ( 1 ) The positions of absorption peaks are indicative of
chemical groups, their ionization states, and bindings to other chemical species of the
nuclei. (2) The intensity of the spectral line is linearly proportional to the number of
nuclei in a particular chemical group. This enables tissue concentrations of metabolites
to be determined by NMR. (3) Where magnetic nuclei are close to other magnetic nuclei,
spectral lines can be assigned to particular chemical groups if two-dimensional NMR
techniques are used (Bax and Lerner, 1986~. In the past decade 31 p NMR spectroscopy
studies on in vivo plant and animal cell cultures have been used to measure the pH of
cell cytoplasm and vacuoles (Roberts et al., 1980; Foyer et al., 1982), phosphate, ATPase
and MgATP activity in root (Roberts et al., 1984) and photosynthetic tissue (Foyer et
al., 1982), and the effects of ammonium (NH4+' vs. nitrate (NO3-' nutrition (Andrade and
Anderson, 1986~. To date, these NMR studies have focused on in vivo tissue cultures,
but there is the promise of live whole-plant analysis. In anticipation, Roberts ( 1984)
outlines numerous studies that could be modified to monitor changes in plant metabolism
as a result of air pollutant exposure. Of particular interest are changes in carbon,
nitrogen and phosphate metabolites in intact leaves, stems, and roots.
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OTHER EMERGING MARKER TECHNOLOGIES
The development of fiber optics, semiconductor and membrane technologies has set
the stage for the development of a variety of microsensors that are capable of taking
physiological/biochemical measurements localized to the level of single cells or implanted
to measure localized tissue parameters (Hirschfeld, 1985~. The driver for this
instrumentation is primarily biomedical research, but there is considerable potential for
use in plant research. IR techniques (Hirschfeld, 1985) have been used quite extensively
in analytical laboratories, and now, as a result of the space program, a method for
physiological measurements has been developed for intact plants. It is claimed that
measurements of such components as chlorophyll, lignin, and protein have developed to
the level of accuracy obtainable by wet chemical analysis.
DISCUSSION
The successful implementation of these new and emerging biological marker
technologies, unfortunately, is usually neither easy nor inexpensive. Sophisticated
instrumentation and computers are required for most of these new technologies, plus
well-trained researchers and technicians to operate them. In the case of SLR, for
example, there is the need for (1) a dedicated accelerator near an environmentally
controlled plant growth facility, (2) sophisticated electronics for tracer profile
measurement, (3) an on-line computing facility for data acquisition and for the analysis
of large data blocks in a relatively short time, (4) advanced and novel mathematical
methods of data analysis and dynamic modeling, and (5) a multidisciplinary approach
coupled with a multidisciplinary team of scientists (Fares et al., 1983~. An NMR facility
requires a similar large investment in facilities, instrumentation and personnel.
The major practical disadvantage to the use of many of these technologies as
biological markers is that, at present, they are of limited use in field studies. So far, all
studies of plant physiology or metabolism using these technologies have been laboratory
studies. The difficulties in implementing these techniques in the field are obvious,
revolving largely around the bulk and delicacy of the instrumentation and the need for a
considerable source of power. Many of these limitations are not insurmountable,
however, and limited field studies using some of these technologies could conceivably be
implemented in the very near future.
The feasibility of bringing 1lC technology to the field has been enhanced by recent
developments in storing high-activity 1lco2 immediately after production (J.D. Goeschl
and C.J. McKinney, pers. comm.). A small, portable, lead-lined pressurized storage
container (whimsically called a "pigs) has been charged with enough 1ico2 so that even
after several hours enough activity remains to conduct a series of viable experiments at
another location. The pig is currently being tested at remote laboratory locations at
Duke University. The next step entails loading detector and gas-monitoring
instrumentation onto a truck with the pig and driving to an experimental field site. The
only remaining requirement is electrical power, which is supplied to most research field
sites anyway. At the field site the gas-delivery cuvettes could be attached to a leaf on
a tree as easily as on one in the laboratory. Thus, the physiological responses of a
plant to air pollutants or other environmental factors could be characterized for field
conditions.
The benefit that balances the cost of establishing and maintaining sophisticated
facilities, either for laboratory or future field studies, is the enormous amount of
invaluable data which can be collected simultaneously over short time intervals. The 1 1C
technique, for example, can make simultaneous, nondestructive, in viva measurements of
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transpiration, stomata! conductance, net photosynthesis both by 12c exchange and 1lc
assimilation, net photosynthate storage in and export from the leaf, turnover time and
pool size of exportable products, axial speed of photosynthate translocation, activity level
of translocated photosynthates, and unloading rate into any given sink tissue. The
significance of these measurements is readily apparent, especially if and when they can
be made in the field. The systems behavior of the overall physiological response may be
and
environmentally induced changes in plant physiology may be evaluated. Genetic and
environmental effects of physiology or growth may be measured. Many of these
parameters cannot be measured through conventional laboratory or field experimental
techniques and those that can usually require months of painstaking replication. When
the amount of information that can be obtained in such a short period from those new
technologies is compared to traditional experimental protocols, these new and emerging
technologies are remarkably cost-effective.
~ ~ ,' ~
characterized. Dynamic carbon allocation patterns may be observed. Genetically
ACKNOWLEDGMENTS
Our carbon-l! studies were made possible by grant No. 86- 11 from the
Quality/Forest Health Program, National Council of the Paper Industry for Air
Stream Improvement. For scientific collaboration and support, we thank Alan A. Lucier
of NCASI; John D. Goeschl, Robert L. Musser, Charles E. Magnuson and Collin J.
McKinney, Phytokinetics Division of PhytoResource Research, Inc.; Boyd R. Strain and the
staff of the Duke University Phytotron;. Walter W. Heck of the Air Pollution Lab,
USDA-ARS, North Carolina State University, and William E. Winner and Richard H.
Waring, Oregon State University. For assistance in outlining the role of NMR, we
thank A. Ian Scott and Howard J. Williams, Center for Biological NMR, Department of
Chemistry, and Ray W. Flumerfelt and Bruce Dale, Department of Chemical Engineering,
Texas A & M University.
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Representative terms from entire chapter:
air pollutants
