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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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82 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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83 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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84 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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85 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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86 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. REFERENCES Andrade, F.H., and I.C. Anderson. 1986. Physiological on corn root tips: A. 13p nuclear magnetic 26:293-296. effects of the form of nitrogen resonance study. Crop Science Bax, A., and L. Lerner. 1986. Two-dimensional nuclear magnetic resonance spectroscopy. Science 232:960-967. Bloom, A.J., F.S. Chaplin, and H.A. Mooney. 1985. Resource limitations in plants: An economic analogy. Ann. Rev. Ecol. Syst. 16:363-392. Fares, Y., D. W. DeMichele, J.D. Goeschl., and D.A. Baltuskonis. 1978. Continuously produced, high specific activity 11c for studies of photosynthesis, transport, and metabolism. Internat. J. Appl. Radiation and Isotopes 29:431-441. Fares, Y., J.D. Goeschl, C.E. Magnuson, B.R. Strain, C.E. Nelson, and H.M.Sadek. 1983. Use of short-lived isotopes in the study of xenobiotic transport. Pp. 364-369 in IUPAC Pesticide Chemistry, Human Welfare and the Environment (J. Muyamoto et al., eds.~. Pergamon Press, New York.

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87 Foyer, C., D. Walker, C. Spenser, and B. Mann. 1982. Observations on the phosphate status and intracellular pH of intact cells, protoplasts and chloroplasts from photosynthetic tissue using phosphorus-31 nuclear magnetic resonance. Biochem. I. 202:429-434. Goeschl, J.D., C.E. Magnuson, Y. Fares, C.H. Jaeger, C.E. Nelson, and B.R. Strain. 1984. Spontaneous and induced blocking and unblocking of phloem transport. Plant, Cell and Environment 7:607-613. Hirschfeld, T., and E.W. Stark. 1984. Near-infrared reflectance analysis of foodstuffs. Pp. 505-551 in Analysis of Food and Beverages (G. Charalambous, em.. Academic Press, Orlando, F1. HirschEeld, T. 1985. Instrumentation in the next decade. Science 230:286-291. Magnuson, C. E., J.D. Goeschl, and Y. Fares. 1986. Experimental tests of the Munch-Horwitz theory of phloem transport: effects of loading rates. Plant, Cell and Environment 9:103-109. McKinney, C.J., Y. Fares, C.E. Magnuson,, C.H. Jaeger, J.D. Goeschl, and J.L. Need. 1988. Automatic system for the control of batch-produced 1 lco2 for continuous labeling experiments. Review of Scientific Instruments 59:467-469. Minchin, P.E.H, and M.R. Thorpe. 1982. Evidence for a flow of water into sieve tubes'associated with phloem loading. J.Exp Botany 33:233-240. Potvin, C., J.D. Goeschl, and B.R. Strain. 1984. Effects of temperature and CO2 enrichment on carbon translocation of plants of the C4 grass species Echinochloa crus-galli (L.) Beauv. from cool to warm environments. Plant Physiol. 75:1054-1057. Potvin, C., B.R. Strain, and J.D..Goeschl. 1985. Low night temperature effect on C4 grass species: II. Effect on photosynthate translocation. Oecologia 67:305-309. Roberts, J.K.M., P.M. Ray, N. Wade-Jardetzky, and O. Jardetzky. 1980.Estimation of cytoplasmic and vacuolar pH in higher plant cells by 31p NMR. Nature 283:870-872. Roberts, I.K.M., D. Wemmer, and O. Jardetzky. 1984. Measurement of mitochondrial ATPase activity in maize root tips by saturation transfer of 31 p nuclear magnetic resonance. Plant Physiol. 74:632-639. Roberts. J.K.M. 1984 Studv c~f nl~nt m~t~holicm in viv~ ,,~ina NMD ct~trmc!~`r , -- ~~ ~ ~-. ~ . ~ ^~~ ~..V~-V1J,. Ann. Rev. Plant Physiol. 35:375-386. Scott, A. I. 1985. Applications of C- 13 NMR in the study of biosynthetic mechanism. J. Natural Products 48:689-707. Sharpe, P.J.H., and H. Wu. 1985. A preliminary model of host susceptibility to bark beetle attack. Pp. 108- 127 in Proceedings, International Union of Forest Research Organizations (L. Safranyik and A.A. Berryman, eds.~. Host Insect Work Group, Banff, Alberta, September, 1983.

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88 Sharpe, P.J.H., H. Wu, R.G. Cates, and J.D. Goeschl. 1985. Energetics of pine defense systems to bark beetle attack. Pp. 206-223 in Proceedings Integrated Pest Management Research Symposium, (S.J. Branham and R.C. Thatcher, eds.) Southern Forest Experiment Station General Technical Report 50-56. New Orleans, Louisiana. Sharpe, P.J.H., and H.W. Scheld, 1986. Role of mechanistic modeling in estimating long-term pollution effects upon natural and man-influenced forest ecosystems. NCASI Technical Bulletin No. 500: Proceedings of Workshop on Controlled Exposure Techniques and Evaluation of Tree Responses to Airborne Chemicals, pp. 72-82. Sharpe, P.J.H., R.D. Spence, and E.J. Rykiel. 1988. Diagnosis of sequential ozone effects on carbon assimilation, translocation and allocation in cottonwood and loblolly pine. NCASI Technical Bulletin (in press).