Stable Isotopes and Plant Carbon-Water Relations

Farquhar

Part I

History and Theoretical Considerations

Introduction to History and Theoretical Considerations

The contributions to this section focus on the fundamentals of plant carbon–water relations, the historical development of stable isotopes in plant biology, and the biochemical basis of fractionation of stable isotopes of carbon, hydrogen, and oxygen. The introductory chapter sets the stage with a summary of basic aspects of gas exchange as they relate to carbon dioxide and water vapor fluxes between plants and the atmosphere and also establishes a common set of terms relating water loss and carbon gain. That chapter is followed by a historical view of stable isotopes in plants by Ehleringer and Vogel, who point out that the initial basic observations on this topic are all derived from geochemistry. O’Leary follows with a discussion of the basic isotope fractionation processes and establishes the linkages between carbon isotopes and plant gas exchange. In the subsequent chapter, Farquhar and Lloyd derive a theory linking plant gas exchange and atmospheric gas composition, illustrating that both carbon and oxygen isotopes in carbon dioxide provide insightful information that can be used to understand physiological processes at the leaf level and then can be used to scale for global considerations. In the last chapter of this section, Flanagan discusses the theory behind how both hydrogen and oxygen stable isotope analyses of leaf water reveal information on biological and atmospheric processes.

1

Introduction: Water Use in Relation to Productivity

Publisher Summary

This chapter discusses the concept of water loss with respect to photosynthesis and provides a set of terms relating to plant-water use. There could be significant differences among cultivars of a crop in water consumption requirements in relation to biomass production. These observations show that atmospheric conditions are of importance in determining absolute relations between water consumption and biomass productivity of a crop. Instantaneous water-use efficiency is defined as the ratio of the fluxes of net photosynthesis (A) and transpiration (E). The advantage of intrinsic water-use efficiency allows direct comparison of intrinsic physiological considerations, factoring out the confounding effects of temperature and humidity gradient differences between plants. While instantaneous and intrinsic water-use efficiencies might be useful in evaluating water and carbon exchange on a short-term basis, they do not necessarily scale to long-term considerations, such as might be of interest to those studying canopy productivity or growth.

I. Introduction

Water loss with respect to photosynthesis has been the topic of numerous books and reviews and has attracted widespread interest from diverse groups, ranging from those interested in basic mechanistic aspects of plant physiology to those concerned with constraints on agricultural production (Fischer and Turner, 1978; Taylor et al., 1983; Sinclair et al., 1984; Stanhill, 1986; Turner, 1986). The intent of this section is to briefly introduce the roots of this interest and to provide a set of terms relating to plant-water use that will be useful when reading the remaining chapters in this volume.

II. A Long-Standing Interest in Water–Use Efficiency

For over a century there has been interest in understanding the relationships between water consumption by plants and overall productivity. Ecologists have been interested in how leaves of different species varied in these parameters, especially in response to seasonal and geographical changes in moisture availability and how these responses influenced both structural and physiological features of natural vegetation. This interest was pioneered by natural scientists and plant geographers (Haberlandt, 1884; Schimper, 1898; Warming, 1909) and later with an ecological emphasis (Livingston and Shreve, 1921). At the same time, agronomists, whose primary goal was increasing productivity, were especially interested in the water requirements for growth of a broad range of crop species (Briggs and Shantz, 1913b).

The pioneering experiments in this area involved growing plants in pots and other containers, where precise (but very time-consuming) information was gained by measuring plant growth and water loss over extended time periods (Briggs and Shantz, 1913a; Shantz and Piemiesel, 1927). From these studies of crop and native plants, it became clear that there was substantial variation in the relationships between water consumption and biomass production. While the mechanistic bases were unknown at the time, plants could be separated into two distinct groups on the basis of their water requirements for growth. We now know that these two groupings represent the C3 and C4 photosynthetic pathways. Furthermore, these early studies established that there could be significant differences among cultivars of a crop in water consumption requirements in relation to biomass production.

These observations showed that atmospheric conditions were of overriding importance in determining absolute relations between water consumption and biomass productivity of a crop. The large site-to-site and year-to-year variations in absolute water consumption characteristics (driven by temperature and humidity conditions) of the same crop were confounding and made it difficult to accept the usefulness of information derived from pot studies (Stanhill, 1986). In part because of the necessity to better understand the climatic factors influencing transpiration and in part because of the need to extend from pot to field studies, micrometeorological aspects predominated from the 1930s through the 1960s (Penman, 1948; Penman and Schofield, 1951; Lemon et al., 1961).

A shift toward appreciating the significance of plants (particularly at the leaf level) in affecting water and carbon fluxes with the atmosphere re-emerged with the pioneering efforts of de Wit (1958). Shortly thereafter, Bierhuizen and Slatyer (1965) published their studies of the interactions between carbon gain through photosynthesis and water loss through transpiration. An analogous situation occurred in the ecological sciences, where the emphasis shifted from understanding community dynamics and structure, which predominated until the early 1960s, to ecophysiological studies of natural systems (Mooney et al., 1987). Today field micrometeorological and physiological approaches are routinely combined in field investigations, and carbon isotope analysis has emerged as a means of spatially and temporally integrating these carbon and water relations parameters (Farquhar et al., 1989).

III. Concepts of Water Use in Relation to Productivity

In evaluating relationships between water loss and growth, a diffuse, sometimes overlapping, and frequently confusing terminology has emerged, which often impedes progress and understanding. In part the differences in terms are driven by different objectives: the interests of a physiologist in understanding mechanisms at the leaf level are not the same as those of an agricultural engineer interested in the marketable crop yield relative to the amount of irrigation applied to a field. Also along the way, terms such as "efficiency’ have emerged, which only add further confusion since they typically do not refer to an efficiency in the same sense as it is understood in engineering. This baggage may not be avoidable in all cases. Nevertheless, useful concepts and terms have emerged to help us better understand the interactions between water and carbon fluxes in evaluating plant physiological responses, growth, and survival.

At the leaf level, carbon and water fluxes involve net photosynthesis (A), measured as CO2 uptake, and transpiration (E), measured as water loss. At steady state these fluxes can be quantified by Ohm’s law analogy relationships:

(1)

and

(2)

where v is the gradient in water–vapor pressure between the leaf and atmosphere divided by total atmospheric pressure, g is the leaf conductance to water vapor, and 1.6 is the ratio of gaseous diffusivities of CO2 and water vapor in air. ca and ci are the external and internal CO2 concentrations. The term v is equal to

(3)

where ei and ea are the water–vapor pressures inside the leaf (assumed to be at saturation) and in the ambient air outside the boundary layer, respectively, and P is the total atmospheric pressure. By expressing the driving gradient for transpiration in this manner, the effects of elevation changes (P) and temperature on diffusion do not have an impact on the estimates of the stomatal conductance (g), which should only be influenced by stomatal pore characteristics (Cowan, 1977). In Eq. (3), the numerator is similar to a term many previous studies have presented as vapor pressure deficit (vpd). The term v has been presented as "Δw" in many previous studies, but such an expression leads to confusion with respect to carbon isotope discrimination (Δ) (sensu Farquhar et al., 1989).

Instantaneous water–use efficiency is defined as the ratio of the fluxes of net photosynthesis (A) and transpiration (E). Since A and E share a diffusion pathway, the stomata, instantaneous water–use efficiency can be determined without an estimate of g:

(4)

Considering the strong role that atmospheric conditions play in determining water–use efficiency through its influence on determining v, many investigators chose to evaluate only the role that biological components play in determining water–carbon exchange relationships. The intrinsic water–use efficiency is defined as A/g, which is really the instantaneous water–use efficiency multiplied by v. However, the advantage of intrinsic water–use efficiency as a term is that it allows direct comparison of intrinsic physiological considerations, factoring out the confounding effects of temperature and humidity gradient differences between plants.

While instantaneous and intrinsic water–use efficiencies might be useful in evaluating water and carbon exchange on a short-term basis, they do not necessarily scale to long-term considerations, such as might be of interest to those studying canopy productivity or growth. Yet the basic notion of assimilation and transpiration can be extended to longer time periods. Here the basic measure is one of the actual biomass produced to the total amount of water consumed in producing that biomass.

Transpiration efficiency (W) is the expression for long-term measures of biomass or yield with respect to water loss at the whole plant level. It can be related to Eq. (4) as

(5)

A fraction of the fixed carbon will be lost through respiration (ϕc) and a fraction of the water (ϕw) may be lost at night if stomata do not completely close. The consequence is that W is not equivalent to A/E. W is defined in terms of molar abundance ratio, yet can just as easily be expressed on a grams-carbon-per-grams-water or grams-biomass-per-grams-water basis. As such, it is particularly important to make clear just exactly which units are being used in the expression.

As will be seen in later chapters in this volume, the carbon isotope composition (¹³C/¹²C) is often used in developing a time-integrated surrogate estimate of either A/E or W. In the strict sense, such an extrapolation is only possible provided the following assumptions are met: (1) the evaporative gradient between leaf and environment (v) is equivalent and known for the species and environments compared and (2) plants and/or species compared do not differ in the fraction of the carbon gain that is lost through respiratory processes (Farquhar and Richards, 1984). Neither of these conditions is likely to be entirely met in a field study of many species. Although carbon isotopic composition values of leaf tissue are unlikely to provide a direct, quantitative comparison of any measure of water–use efficiency, they should provide a relative index for ranking A/E or W among species. Furthermore, calculations can be made to determine the extent to which errors arising from differences in leaf temperatures among plants being compared affect interpretation of carbon isotope composition data.

Frequently, agronomists are not so much interested in W but in the crop yield (biomass of harvestable product such as seed) per unit of water applied to the crop. This expression is not equal to W but is exactly equal to the product of W and the harvest index.

The expression W also lacks consideration of evaporative moisture loss occurring from the soil surface. Such losses can occur early in the growing cycle of a crop or because inadequate moisture levels limit growth and prevent complete canopy cover of the soil surface. Of importance to the agronomists will be a measure that incorporates the transpiration efficiency to the fraction of water actually consumed by the crop. Crop water–use efficiency (C) is defined as

(5)

where E is the transpirational water loss through the crop and Etotal is the total water loss from the soil (Passioura, 1977). Both water–loss rates are integrated over the life of the crop and expressed on a unit ground area basis.

None of the above "efficiency’ terms are clearly measures of efficiency as typically considered in engineering, although the last definition does come close since it measures the extent to which the plant is able to process the moisture loss from the soil.

Alternatively, we could express relationships between carbon and water fluxes in an inverse manner. That is, instead of A/E, we could express the patterns through E/A. Such was actually the approach used in the pioneering efforts at the beginning of this century. The "water requirement’ was defined as the ratio of the amount of water transpired through the plant relative to the amount of biomass produced. Mathematically, the water requirement would be equivalent to 1/W. Today the expression "water requirement’ is not used. However, in its place, it is common in the literature to find the term transpiration ratio.

References

Bierhuizen, J.F., Slatyer, R.O. Effects of atmospheric concentration of water vapour and CO2 in determining transpiration-photosynthesis relationships of cotton leaves. Agric. Meteor. 1965; 2:259–270.

Briggs, L. J., and H. L. Shantz. 1913a. The water requirements of plants. I. Investigations in the Great Plains in 1910 and 1911. USDA Bureau of Plant Industry, Bulletin 284.

Briggs, L.J., Shantz, H.L. The water requirement of plants. II. A review of the literature. Bulletin: USDA Bureau of Plant Industry, 1913; 285.

Cowan, I.R. Stomatal behavior and environment. Adv. Bot. Res. 1977; 4:117–227.

de Wit, C. T. 1958. Transpiration and Crop Yields. Institute of Biological and Chemical Research on Field Crops and Herbage. Wageningen, The Netherlands.

Farquhar, G.D., Richards, R.A. Isotopic composition of plant carbon correlates with water–use efficiency of wheat genotypes. Aust. J. Plant Physiol. 1984; 11:539–552.

Farquhar, G.D., Ehleringer, J.R., Hubick, K.T. Carbon isotope discrimination and photosynthesis. Annu. Rev. Plant Physiol. Mol. Biol. 1989; 40:503–537.

Fischer, R.A., Turner, N.C. Plant productivity in the arid and semi-arid zones. Annu. Rev. Plant Physiol. 1978; 29:277–317.

Haberlandt, G.Physiologische Pflanzenanatomie.. Leipzig: Verlag von Wilhelm Englemann, 1884.

Lemon, E.R., Stewart, D.W., Shawcroft, R.W. The sun’s work in a cornfield. Science. 1961; 174:371–378.

Livingston, B.E., Shreve, F.The Distribution of Vegetation in the United States, as Related to Climatic Conditions.. Washington, D.C.: Carnegie Institution of Washington, 1921.

Mooney, H.A., Pearcy, R.W., Ehleringer, J.R. Plant physiological ecology today. BioScience. 1987; 37:18–20.

Passioura, J.B. Grain, yield, harvest index, and water use of wheat. J. Aust. Inst. Agri. Sci. 1977; 43:117–120.

Penman, H.L. Natural evaporation from open water, bare soil, and grass. Proc. R. Soc. London A. 1948; 193:120–146.

Penman, H.L., Schofield, R.K. Some physical aspects of assimilation and transpiration. Sym. Soc. Exp. Biol. 1951; 5:115–129.

Schimper, A.F.W.Pflanzen-Geographie auf Physiolgischer Grundlage.. Jena: Fisher, 1898.

Shantz, H.L., Piemeisel, L.N. The water requirement of plants at Akron, Colorado. J. Agri. Res. 1927; 34:1093–1190.

Sinclair, T.R., Tanner, C.B., Bennett, J.M. Water–use efficiency in crop production. BioScience. 1984; 34:36–40.

Stanhill, G. Water use efficiency. Adv. Agron. 1986; 39:53–85.

Taylor, H.M., Jordan, W.R., Sinclair, T.R.Limitations to Efficient Water Use in Crop Production.. Madison, WI: American Society of Agronomy, 1983.

Turner, N.C. Crop water deficits: A decade of progress. Adv. Agron. 1986; 39:1–51.

Warming, E.Oecology of Plants: An Introduction to the Study of Plant Communities.. Oxford, U.K.: Clarendon Press, 1909.

2

Historical Aspects of Stable Isotopes in Plant Carbon and Water Relations

James R. Ehleringer and John C. Vogel

Publisher Summary

This chapter discusses the historical aspects of stable isotopes in plant carbon and water relations. The linkage between ¹³C content and photosynthetic pathway type make it possible to determine the extent of C4 photosynthesis within the plant kingdom. The chapter discusses the basis of ¹³C variation in plants. Applications of leaf ¹³C content to ecophysiology have arisen out of the observation that the isotopic depletion in plants is influenced by environmental factors. There was limited attention by plant physiologists to leaf-water evaporative enrichment until recently when models linking carbon isotope fractionation and transpiration efficiency became better understood. One goal of renewed interest has been to determine whether isotopic evaporative enrichment of leaf water can be used as a direct measure of the leaf-to-air water vapor gradient and if so, this would make it possible to calculate the absolute estimates of transpiration efficiency through combined studies of the hydrogen and oxygen isotopes of leaf water and the carbon isotope ratios of leaves. This effect should be especially useful for assessing the degree to which plants can limit water loss under hot and dry conditions in their natural environment.

Most of the history of stable isotopes in plant biological research began outside of biology. Interest in stable isotopes first developed in the physical sciences in the mid-1930s and then by the 1940s became a major component of geology. Only in the past 2 decades have plant biologists shown widespread interest in stable isotopes. The original botanical contributions in the area of stable isotopes are largely a history of contributions by geochemists with very broad interests who wanted to know more about natural variations in isotopic abundance levels. In this brief chapter, we attempt to reconstruct some of that history, specifically as it relates to plant water and carbon relations, and to describe the hows and whys of the development of botanical interests in stable isotopes.

Harold Urey and co-workers discovered a heavy form of hydrogen, deuterium, in the early 1930s. Once deuterium was known, it was only natural to investigate whether there was variation in the abundance of light and heavy isotopic forms of hydrogen in nature. Careful measurements of the density of water revealed considerable differences in the heavy isotope content of samples of different origin. While this approach was satisfactory for looking at heavy water, it was not practical for studying the abundance of isotopic forms of other elements that were being discovered during this period. A major breakthrough came when Nier and colleagues developed the modern isotope mass spectrometer (Nier, 1936, 1940), which allowed precise measurements of the relative abundances of light and heavy isotopic forms of hydrogen as well as other elements, such as carbon and oxygen. The development of mass spectrometers of sufficiently high precision in the late 1940s prompted systematic surveys of the variability in the isotopic composition of the light elements (H, C, N, O) in nature. These studies were notably undertaken in the laboratory of Harold Urey at the University of Chicago by Harmon Craig (¹³C), Sam Epstein (¹⁸O), Irving Friedman (²H), Cesare Emiliani (¹⁸O thermometry), and other co-workers (see review by Nier, 1990).

I. First Observations of Carbon Isotope Fractionation in Plants

Using a mass spectrometer, Nier and Gulbransen (1939) were the first to observe that the heavy isotope of carbon, ¹³C, was slightly depleted in plants with respect to inorganic carbonaceous materials such as limestone. Shortly thereafter, Murphy and Nier (1941) went on to show that there was variation in ¹³C content in wood among different plant species.

The early surveys of the ¹³C/¹²C ratio of carbon in plants (Wickman, 1952; Craig, 1953) were accompanied by attempts to explain the observed ¹³C depletion. Based on herbarium samples, Wickman (1952), working in Stockholm, observed that plants collected along railroad stops from central Asian deserts were more enriched in ¹³C than plants from tropical rain forests. He suggested that there was a cyclic process in which the degree of depletion of ¹³C in plants depended on the amount of recycling of soil-derived carbon dioxide. Craig (1954) pointed out, however, that this mechanism could not explain the observed fractionation satisfactorily. Harmon Craig favored an alternative hypothesis—that fractionation processes within leaves accounted for the variation in ¹³C composition and that perhaps the environment played a role in influencing the magnitude of these fractionations. Meanwhile Baertschi (1953), working in Basel, showed in laboratory experiments that no fractionation occurred during the respiration of bean seedlings and that ¹³C was depleted by about 26‰ during CO2 assimilation/photosynthesis. The similarity of this fractionation factor with that found by Urey in 1948 (29‰) and by Calvin and Weigl in 1952 (27‰) for algae (cited by Baertschi, 1953) suggested to him that the fractionation process was basically the same for different plants and growth conditions. The values these authors obtained, incidentally, correspond closely to the maximum fractionation factor for photosynthesis observed in nature.

Craig’s (1954) in-depth discussion of the biological fractionation process formed the basis for further experimental work by Park and Epstein (1960, 1961). Rod Park and Sam Epstein proposed a three-step model to account for the observed differences in ¹³C composition of leaves from the CO2 in the atmosphere. Diffusion and photosynthetic fractionation were important components of their model, with secondary plant metabolism accounting for only a small additional fractionation. They correctly reasoned that leaf-level fractionation must occur, since the isotopic composition of dissolved CO2 was not the same as that of the carbohydrate formed. Park and Epstein suspected that translocation of enriched ¹³CO2 to the root system played an important part in determining the overall fractionation. At the time, there was supportive evidence from other investigators, showing that roots evolved CO2 at high rates during the light. However, Park and Epstein (1960, 1961) were unable to confirm that the root CO2 was enriched in ¹³C. Here the quest to understand the fractionation process in plants rested for nearly 20 years.

II. Discovery of C4 Photosynthesis

Without it yet being realized, C4 plants had been represented in the first plant surveys: Wickman (1952) found four desert specimens with abnormally small discrimination against ¹³C (Calligonum acanthopterum in the Polygonaceae and Arthrophytum arborescens, Haloxylon aphyllum, and Salsola richteri in the Chenopodiaceae). Since these plants derived from the central Asian deserts with high-carbonate soils, they formed an important aspect of Wickman’s cyclic enrichment model. Wickman (1952) also found one emergent aquatic grass (Paspalum distichum) with high ¹³C content. It is perhaps just one of these odd facts that this grass was collected at Lake Laugunita on the Stanford University campus, where 20 years later the Carnegie group on that campus would conduct their pioneering research on the genetics and physiology of C4 photosynthesis.

Craig (1953) also had one unknown grass from southwestern Kansas that had high ¹³C content, while all other plant samples collected from that pedocalcic site had normal values. He reexamined that grass sample after washing it with HCl to ensure that any carbonates would be removed. When he observed the same ¹³C content as before, Harmon Craig concluded that the sample has either utilized bicarbonate or carbonate ion directly in its photosynthesis, or that it has used carbon dioxide derived from the bicarbonate ion. Unfortunately, Craig was incorrectly referring to the possibility that CO2 from calcium carbonate at the soil surface was serving as the photosynthetic CO2 source for this grass (because either the air was enriched or there was direct uptake through the roots).

The connection between the C4 syndrome and the ¹³C content in terrestrial plants only became clear after the discovery of the C4 pathway in tropical grasses by Kortshak et al. (1965) and Hatch and Slack (1966). Margaret Bender (1968) published the first ¹³C data showing an association between enriched isotopic composition and the newly discovered photosynthetic pathway. Extension of this anomalous group of plants to other families soon followed (Bender, 1971; Smith and Epstein, 1971), and it was not long before isotopic studies led to the discovery that C4 species were found to belong to at least 13 different families, suggesting parallel evolution rather than one common origin. The fact that certain succulents showed anomalous ¹³C values was also noted at this time (Vogel and Lerman, 1969; Bender, 1971) and was soon linked to crassulacean acid metabolism (Bender et al., 1973).

III. Carbon Isotopes and Photosynthetic Pathway Distribution

The linkage between ¹³C content and photosynthetic pathway type make it possible to more easily determine the extent of C4 photosynthesis within the plant kingdom. The late 1960s and 1970s were an exciting time for tracing the phylogenetic distribution of C3, C4, and CAM photosynthesis among different plant taxa. The charge was lead by two distinct groups: established geochemists/physicists broadly interested in botany (such as Margaret Bender in Wisconsin and John Vogel in South Africa) and a group of plant physiologists who were either doing postdoctoral research in geochemical laboratories or had access to necessary instrumentation. Among the pioneering group of plant physiologists were Bruce Smith working in Texas; Bruce Tregunna, John Downton, and Joe Berry in Canada; John Troughton in New Zealand; Barry Osmond in Australia; Elien Deleens in France; and Klaus Winter and Hubert Ziegler in Germany. The 1969 U.S.–Australia meeting on photosynthesis and photorespiration in Canberra (Hatch et al., 1971) served as a focal point for what was known at the time about carbon isotope composition and photosynthesis (Downton, 1971). The isotopic data base expanded rapidly during the 1970s and Downton (1975) and Raghavendra and Das (1976, 1978) produced summary lists of the families known to have C4 taxa, pointing out that a number of the genera contained both C3 and C4 species.

Later studies and field surveys continued to fill gaps in our knowledge of the taxonomic distribution of C3 and C4 photosynthesis. One of the basic observations that emerged from these field studies was that there could be substantial variation in the ¹³C content of C3 photosynthesis, even within a species, while much less variation occurred among C4 plants (O’Leary, 1988). While it was presumed that this isotopic variation encompassed both environmental and genetic components, the basis contribution of each component remained unclear until the basis of the fractionation events were elucidated.

IV. Understanding the Basis of ¹³C Variation in Plants

Once detailed descriptions of the kinetic isotope fractionation process during photosynthesis were produced in the late 1970s (Vogel, 1980; O’Leary and Osmond, 1980; O’Leary, 1981; Farquhar et al., 1982) the scene was set for the development of direct application of carbon isotope studies to physiological processes. It was especially Graham Farquhar, Marion O’Leary, and Joe Berry (1982) who showed that the carbon isotope ratio of an individual plant correlated with intercellular carbon dioxide levels and that this could be used in the selective breeding of high transpiration efficiency genotypes (Farquhar and Richards, 1984). Several chapters in this volume deal with further developments in this field.

Applications of leaf ¹³C content to ecophysiology have also arisen out of the observation that the isotopic depletion in plants is influenced by environmental factors. One such application is related to the original cyclic depletion model proposed by Wickman (1952) and by atmospheric CO2 observations of Keeling (1958); it can be observed in dense forests where recycling of respired carbon dioxide takes place—the so-called canopy effect (Vogel, 1978). While the phenomenon is somewhat more complicated than originally proposed, it seems clear that isotopic variation within canopies reflects both a recycling of respired carbon dioxide as well as increased intercellular carbon dioxide levels in the lower leaves of the canopy (Schleser and Jayasekera, 1985; Ehleringer et al., 1986; Sternberg et al., 1989). Several chapters in this volume deal with the current efforts to use ¹³C composition of plants to address recycling of carbon within forest canopies.

V. Water and Evaporative Enrichment

Studies of hydrogen and oxygen isotope ratios are quickly assuming greater importance in plant biology, but again the history of research on these elements began outside of biology. Early workers in the 1950s in Europe and the United States measured the ¹⁸O and ²H content of different freshwater samples (Epstein and Mayeda, 1953; Friedman, 1953; Dansgaard, 1954), showing that fresh water typically contained less of the heavy isotopes than did seawater. However, it was the systematic investigations of Dansgaard (1961, 1964) in Denmark that provided the framework for a mechanistic understanding of the factors contributing to the geographic isotopic variation in meteoric waters.

Harold Urey (1947) calculated that atmospheric oxygen at equilibrium should be enriched in ¹⁸O by 6‰ relative to seawater. Yet when Dole et al. (1954) measured atmospheric diatomic oxygen, they observed an ¹⁸O excess of 23‰. Since this diatomic oxygen originates from photosynthesis and because evaporative processes were known to result in an enrichment of surface waters (Craig and Gordon, 1965), early investigations began to analyze the isotopic composition of leaf water which becomes enriched during transpiration (Gonfiantini et al., 1965). The extent of the leaf-water evaporative enrichment could be modeled (Dongmann et al., 1974; Zundel et al., 1978), and one of the key parameters influencing enrichment of the heavier isotopes was relative humidity. Early in these efforts, Farris and Strain (1978) suggested that water stress could directly impact the extent of leaf-water enrichment. However, it now appears that a significant amount of the isotopic enrichment by water-stressed leaves may be the result of elevated leaf temperatures in stressed plants (Flanagan and Ehleringer, 1991).

There was limited attention by plant physiologists to leaf-water evaporative enrichment until recently when models linking carbon isotope fractionation and transpiration efficiency became better understood (see recent progress detailed by Farquhar et al., Chapter 5; Flanagan, Chapter 6; and Yakir et al., Chapter 33, in this volume). One ultimate goal of the renewed interest has been to determine whether or not isotopic evaporative enrichment of leaf water can be used as a direct measure of the leaf-to-air water vapor gradient, the driving force for transpiration. If so, this would make it possible to calculate absolute estimates of transpiration efficiency through combined studies of the hydrogen and oxygen isotopes of leaf water and the carbon isotope ratios of leaves. This effect should be especially useful for assessing the degree to which plants can limit water loss under hot, dry conditions in their natural environment.

VI. Environmental Signals in D/H and ¹⁸O/¹⁶O of Plant Tissues

The investigation of deuterium (²H or D) in plant tissues was initiated by a general survey of deuterium in organic matter in the early 1970s (Schiegl, 1970, 1972; Schiegl and Vogel, 1970; Epstein et al., 1976). These studies showed that the hydrogen in the tissue of plants is considerably depleted in deuterium with respect to the source water from which it was derived, viz the leaf sap or, in the case of hydrophytes, the aqueous substrate. Schiegel (1970) found that there was a correlation between D/H ratio and climate, with D/H ratio strongly correlated with temperature. Early in these studies, Sam Epstein and colleagues at Cal Tech focused on the D/H ratio of organic tissues as paleorecorders (Epstein et al., 1976; Yapp and Epstein, 1982). In particular, they concentrated on cellulose in wood, since this material is laid down during a single season, clearly distinguishable as tree rings within the wood, and not modified thereafter. Recognizing that the hydrogen in hydroxyl groups of cellulose could continue to exchange with water within the plant, Epstein and colleagues restricted their analyses to only the D/H ratio of C—H groups in cellulose (Epstein et al., 1976).

Physiological studies were soon to follow. Ziegler et al. (1976), Sternberg and De Niro (1983), and Leaney et al. (1985) showed that there were distinct differences in D/H ratio among C3, C4, and CAM plants, while Epstein and co-workers started to record the differences in isotopic composition of various chemical substances in the plant (Smith and Epstein, 1970; Epstein et al., 1976, 1977; Lenhart, 1979; Ziegler, 1979).

Since techniques were developed for measuring the ¹⁸O content of organic matter (e.g., Hardcastle and Friedman, 1974), this isotope has been analyzed together with deuterium in studies of the fractionation processes within the plant. Since oxygen is potentially derived not only from water but also from carbon dioxide, it was thought that the ¹⁸O content of organic matter could conceivably show a different pattern from that of the D/H ratio. De Niro and Epstein (1979), however, established that equilibration with aqueous phase is established before ¹⁸O incorporation in the tissue, so that the initial ¹⁸O/¹⁶O ratio is determined by the plant water alone.

The application of ²H and ¹⁸O analyses of plant tissue in ecological studies is actively being pursued (cf. Ziegler 1989; Sternberg 1989; White 1989) and will undoubtedly produce many interesting results in the near future. Some of these topics are discussed later by Farquhar and Lloyd (Chapter 5), by Flanagan (Chapter 6), and also by Yakir and colleagues (Chapter 33).

VII. Water Movement into Plants

At the same time as the leaf-water studies were being initiated, Gonfiantini et al. (1965) and Wershaw et al. (1966) showed that there was no hydrogen or oxygen isotope fractionation during water uptake through roots. These observations established that xylem sap analyses could be used for determining plant-water sources by comparing the nonenriched moisture in the stems of plants with that of the possible sources. White et al. (1985) and Sternberg and Swart (1987) were among the first to apply these principles to ecological studies; their results showed that adjacent plants could use different water sources and that plants could shift between sources over short time periods. This application is now gaining widespread interest because of its power in quantitatively assessing belowground activities. Later in this volume, Dawson (Chapter 30) and Thorburn and Walker (Chapter 32) examine the recent progress in this area.

References

Baertschi, P. Die Fraktionierung der natürlichen Kohlenstoffisotopen im Kohlendi-oxydstoffwechsel grüner Pflanzen. Helv. Chim. Acta. 1953; 36:773–781.

Bender, M.M. Mass spectrometric studies of carbon-13 variations in corn and other grasses. Radiocarbon. 1968; 10:468–472.

Bender, M.M. Variations in the ¹³C/¹²C rations of plants in relation to the pathway of photosynthetic carbon dioxide fixation. Phytochemistry. 1971; 10:1239–1244.

Bender, M.M., Rouhani, I., Vines, H.M., Black, J.C.C. ¹³C/¹²C ratio changes in crassulacean acid metabolism plants. Plant Physiol. 1973; 52:427–430.

Craig, H. The geochemistry of the stable carbon isotopes. Geochim. Cosmochim. Acta. 1953; 3:53–92.

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