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Carbon Isotope Discrimination as a Tool to Screen for Improved Drought Tolerance

Bert Cregg1 and Jianwei Zhang2

1. Department of Horticulture, Michigan State University, East Lansing, MI 48824 USA
2. Westvaco Forest Research, Wickliffe, KY 42087 USA

A paper from the Proceedings of the 11th Metropolitan Tree Improvement Alliance (METRIA) Conference held in Gresham, Oregon, August 23-24, 2000, and cosponsored by the Landscape Plant Development Center.

Abstract

The isotopic ratio of 13C to 12C in plant tissue is less than the isotopic ratio of 13C to 12C in the atmosphere, indicating that plants discriminate against 13C during photosynthesis. Variation in discrimination against 13C during photosynthesis is due to both stomatal limitations and enzymatic processes. Theoretical and empirical studies have demonstrated that carbon isotope discrimination is highly correlated with plant water use efficiency. Analysis of carbon isotope discrimination has conceptual and practical advantages over measuring water use efficiency by instantaneous measurements of gas exchange or whole-plant harvests. Carbon isotope discrimination provides an integrated measure of water-use efficiency, samples are easily collected, and processed, and large numbers of samples may be collected in diverse environments. Moreover, in woody plants, carbon isotope discrimination can be determined on annual ring samples, providing a historical analysis of plant response to environmental conditions. In this paper we present results from several studies on genetic variation in carbon isotope discrimination and water use efficiency in trees, from our past research and from the literature. We discuss application of carbon isotope analysis in tree improvement programs as well as potential limitations of the technique.

Introduction

Water is commonly the most limiting environmental factor for tree Survival and growth. Water may be especially limiting in urban environments where limited soil volumes, soil compaction, and elevated temperatures can combine to increase tree moisture stress (Bassuk and Whitlow 1985, Clark and Kjelgren 1990, Whitlow at al. 1992, Cregg 1994, Cregg and Dix 2000). Urban foresters, like all foresters, have two principle sets of management tools; culture and selection. Cultural options to minimize the impact of drought stress on tree survival and growth are varied and include irrigation, increasing planter size, mulching, and the use of prescription soil mixes. However, cultural treatments may not be affordable or practical in many instances. Therefore, selecting trees for improved drought tolerance may represent the best option to improve urban tree survival and growth on a large scale.

Genetic variation in the physiological mechanisms associated with drought tolerance has been studied in several tree species, including loblolly pine (Pinus taeda L.) (van Buijtenen et al. 1976, Bongarten and Teskey 1986, Bongarten and Boltz 1986, Teskey et al. 1987, Seiler and Johnsen 1988), black spruce (Picea mariana [Mill.] B.S.P.) (Bernier et al. 1994, Johnsen and Seiler 1996, Johnsen et al. 1996), and ponderosa pine (Pinus ponderosa Dougl. ex Laws.) (Monson and Grant 1989, Cregg 1993a, 1994). These studies indicate significant, and potentially exploitable, intra-specific variation in a number of morpho-physiological traits related to drought tolerance including rate of gas exchange, osmotic adjustment, and stomatal density. Moreover, several recent studies have demonstrated carbon isotope discrimination (D) may be used as a surrogate to select for improved water-use efficiency in crops and trees (Farquhar et al. 1989). In the following discussion we will describe the theory of carbon isotope discrimination and how it relates to water use efficiency. Some of the pitfalls and limitations associated with applying the technique as a screen for improving drought tolerance will be described. We will conclude by describing how selection for D may be incorporated in to an urban tree improvement program.

Theory

The isotopic ratio of 13C to 12C in plant tissue is less than the isotopic ratio of 13C to 12C in the atmosphere, indicating that plants discriminate against 13C during photosynthesis. The isotopic ratio of 13C to 12C in C3 plants (d13C) varies mainly due to discrimination during diffusion and enzymatic processes. The rate of diffusion of 13CO2 across the stomatal pore is lower than that of 12CO2 by a factor of 4.4‰. Additionally, there is an isotope effect caused by the preference of ribulose bisphosphate carboxylase (Rubisco) for 12CO2 over 13CO2 (by a factor of ~27‰). In both cases, the processes discriminate against the heavier isotope, 13C (Farquhar et al. 1989). Based on the work of Farquhar the linkage between discrimination against 13C during photosynthesis and water use efficiency may be demonstrated by the following relationships. The stable isotope ratio (d13C) is expressed as the 13C/12C ratio relative to a standard (PeeDee Belemnite) (Craig 1957). The resulting d13C value may be used to estimate isotope discrimination (D) as:

D= (da – dp)/(1+ dp)

Where dp is the isotopic composition of the plant material and da is that of the air (assumed to be 8‰). As CO2 assimilation (A) increases or stomatal conductance (gs) decreases, intercellar CO2 decreases resulting in decreased discrimination against 13C. The relationship between ci and D is represented by the model of Farquhar et al (1982):

D = 4.4 + 22.6(ci/ca)

Where ci is the intercellular CO2 and ca is atmospheric CO2 ( ≈ 355 ppm).

The amount of isotopic discrimination that occurs during assimilation may be compared by D or d13C. Carbon isotope discrimination (D) may be intuitively easier to grasp but cannot be calculated if atmospheric d13C is not known or cannot be assumed to be equal to ambient (e.g., growth chamber experiments).

Empirical Relationships Between D and WUE

Water use efficiency may be estimated from measurements of dry weight accumulation over time relative to amount of water transpired (transpiration efficiency, TE) or by measurements of gas exchange (instantaneous water use efficiency, WUEi). Instantaneous WUE may be calculated as the ratio of assimilation to stomatal conductance or transpiration (A/gs or A/E). Because E is a function of both gs and vapor pressure deficit, A/g is sometimes referred to as intrinsic water use efficiency. Based on the relationships described above, D is linked to WUEi through the effects of A and gs on ci. As WUEi increases due to stomatal closure (decrease gs) or an increase in A, intercellular CO2 declines and discrimination decreases. Therefore, WUEi is inversely related to D and positively related to dC13.

A strong correlation between D or d13C and ci/ca or WUEi has been reported for numerous crop and tree species. Johnson et al. (1993) reported that correlations between D and A/g ranged between –0.77 and –0.91 for crested wheatgrass in a series of greenhouse and field studies. In the same trials the correlation between D and transpiration efficiency ranged between –0.73 and –0.94. In a study of western larch (Larix occidentalis Nutt.) seedlings, Zhang and Marshall (1994) found that D was significantly (P<0.0001) correlated with transpiration efficiency (r= -0.85) and instantaneous water use efficiency (r = -0.70).

Genetic Variation in D

The correlation between water use efficiency and D has been extensively studied in several crops including common bean (Phaseolus vulgaris L.) (Ehleringer 1990, Ehleringer et al. 1991), wheat (Triticum aestivum L.) (Farquhar and Richards 1984 and Condon et al. 1990), peanut (Arachis hypogea L.) (Hubick et al. 1986 and Wright et al. 1994), barley (Hordeum vulgare L.) (Acevedo 1993), and cowpea (Vigna unguiculata [L.] Walp.) (Ismail et al. 1994). These studies suggest that genetic variation in D may be sufficient to be useful as a selection criterion for improved water use efficiency.

The extent of intra-specific variation in D of forest trees has been analyzed only in a few species including Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco) (Zhang et al. 1993), black spruce (Flanagan and Johnsen 1995), boxelder (Acer negundo L.) (Dawson and Ehleringer 1993), and western larch (Larix occidentalis Nutt.) (Zhang and Marshall 1994, Zhang et al. 1994). Zhang and Cregg (1996) observed significant intra-species variation in D in several Pinus species including P. ponderosa. Cregg and Zhang (In press) recently studied the carbon isotopic composition of Scots pine (Pinus sylvestris L) seedlings from diverse seed sources representing a longitudinal transect across the range of the species. They found significant variation among seed sources. Moreover, the dC13 was significantly correlated with precipitation of the seed source, indicating Siberian seed sources had higher water efficiency than European seed sources

Advantages of D as a Selection Criteria for improved WUE

Carbon isotope discrimination has several conceptual and logistical advantages to screening for drought tolerance based on TE or WUEi. Carbon isotope discrimination integrates ci/ca over the time the sampled tissue was formed. In contrast, WUEi measured by gas exchange provides ‘snapshots’ of A/g or A/E and may not be representative of overall WUE. Measurements of D are much less time and labor intensive than calculation of whole plant water use and dry weight data needed to calculate TE.

One particular advantage of using isotope analysis in trees is that isotope discrimination can be determined on annual rings from increment cores (Livingston and Spittlehouse 1993, MacFarlane et al. 1999). Thus, D or d13C can be determined across the range of climatic conditions that may have occurred over the life of the tree (e.g., drought versus wet years). In addition to sampling across years using increment cores, the effect of varying weather patterns on isotope discrimination can be assessed on evergreen trees that retain multiple year’s of leaves. For example, in a study of ponderosa pine growing in two range-wide provenance tests, Cregg et al. (2000) determined D of needles formed in four years in one location and three years at the second location. Age:age correlations are generally high for isotope discrimination indicating a high degree of reproducibility in values and low genotype x environment (G x E) interactions associated with variation in precipitation (Hall et al. 1994). Zhang and Cregg (In preparation for Tree Physiol.) found consistent and significant (P<0.0001) year: year correlations among six years’ foliage samples and among five years’ increment core samples from 10 ponderosa pine seed sources grown in range-wide provenance tests in Nebraska. Age:age correlations in leaf D of Picea mariana trees varied from 0.70 to 0.84 (Johnsen 1999). Cregg et al.(2000) analyzed G x E interaction in D among four seed sources of ponderosa pine grown in Oklahoma and Nebraska. They found that within a location, year x source interactions were non-significant (P>0.05), indicating that seed source rankings were stable across years of varying rainfall.

Recent evidence suggests that D may be also correlated with productivity. Height growth of ponderosa pine seed sources was significantly (P<0.05, r=0-81) correlated with D, indicating that sources with increased water use efficiency grew faster. Johnsen et al. (1999) found an extremely tight relationship (r=-0.97) between breeding values for tree height and D in black spruce. The negative relationship between discrimination values and growth suggests that genetic variation D is attributable to variation in photosynthetic capacity.

Potential Pitfalls and Limitations

While the use of isotope discrimination clearly has advantages over other assessments of water use efficiency, there are several factors that need to be considered in evaluating its use in a selection program.

Cost

The cost of carbon isotope sampling varies depending up the laboratory, the level of processing, and type of sample. Some laboratories vary their fees depending on the type of organization, giving a discount to universities and other non-profit agencies. In general, costs range from $15 to $60 with an average cost for non-profits around $20 for standard oven-dried and ground tissue. Thus, sampling for a relatively simple experiment comparing 10 genotypes with 10 trees per genotype would cost roughly $2000 (10 genotypes x 10 trees x $20). If the investigator increases replication, the number of genotypes sampled, types of tissues sampled, years sampled, locations sampled, and so on, the costs can multiply rapidly.

Relevance of WUE as a drought tolerance mechanism

All other factors being equal, genotypes with high water use efficiency will survive and grow better in water-limiting environments than genotypes with low water use efficiency. However, in nature all other factors are rarely equal. The physiological basis for variation in drought tolerance in a given tree species may be due to a wide and potentially unrelated array of mechanisms including needle morphology, allocation patterns, gas exchange patters, osmotic adjustment, and hydraulic architecture. In general, selection for improved water use efficiency through analysis of carbon isotopes will be most useful in selection for maintenance of growth under drought rather than survival. Survival mechanisms may relate more to growth phenology and allocation patterns than improved carbon gain per unit water loss (Cregg 1993). For example, in comparing ponderosa pine populations that were known to vary in survival under imposed drought, Zhang et al. 1997 found that variation WUEi and d13C was minimal. In these populations increased survival under imposed drought was more strongly related to allocation to roots than to gas exchange characteristics (Cregg 1994). Pennington et al. (1999) found substantial genetic variation d13C of honey mesquite (Prosopis glandulosa Torr.) but determined that a drought escape mechanism was most important for growth and survival under drought for the species. Mesquite seed sources adapted to the driest region of the species range had relatively quick seed emergence and completed growth when soil moisture was adequate.

Importance of phenology

As mentioned previously, plant phenology or the timing of growth can play a role in interpreting carbon isotope data. Cregg et al. (2000) compared D values among four diverse seed sources of ponderosa pine grown at two locations in the Great Plains; Plattsmouth, NE and Norman, OK. Analysis of growth patterns among the seed sources indicated significant differences in phenology. The southernmost seed source (New Mexico) broke bud later and continued shoot and needle growth much later in the growing season than the other sources. Thus, the carbon isotope signature of the NM seed source foliage was formed under a different range of environmental conditions than foliage the other seed sources. Moreover, there were significant changes in growth patterns for one source (Nebraska) between the two locations. This seed source also showed the most plastic response in D between the two locations.

Hydraulic conductivity and branch length

Several recent investigations (Panek and Waring, 1995, Panek 1996, Walcroft et al. 1996, Warren and Adams 2000) have demonstrated the importance of branch length and hydraulic conductivity in determining the carbon isotope signature in the foliage of trees. Warren and Adams (2000) found that branch length was the principle determinant of variation in d13C in Pinus pinaster Ait. trees. Variation in d13C was also highly correlated with hydraulic conductivity. Stable isotope composition was inversely related to leaf specific hydraulic conductivity of Douglas-fir trees across a climate gradient in Oregon (Panek 1996). Isotope discrimination is related to hydraulic conductivity because stomata close in response to increasing tension in the xylem (Irvine et al. 1999).

Variation in d13C within in the canopy

Re-fixing of respired carbon can affect the carbon isotope signal of understory foliage. In forest stands, CO2 concentrations increase near the ground due to efflux of soil respired CO2. The isotopic composition of respired air differs form the bulk atmosphere. The extent to which vertical gradients of d13C exist in foliage depend on a number of stand and climatic factors (Buchman 1998). The greater the mixing and turbulence within the stand the less opportunity for gradients to develop. Open stands or stands in exposed areas will have greater air mixing and less likely to have d13C gradients. Brooks et al. estimated that 20% of the variation in d13C gradient in a canopy of boreal conifers was due to variation in the isotopic composition of the source air.

Role of D in an urban tree selection program

From the foregoing discussion we may conclude the following. The carbon isotope composition of plant tissue:

These factors make isotope analysis attractive to those interested in selecting genotypes for improved stress tolerance. The advantages must be balanced against some potential limitations, which include:

In order to use 13C sampling effectively in a program to select trees for stressful sites we suggest the following:

Identify the role of improving water use efficiency in improving drought tolerance of the species in question and its use in the landscape. This will likely require subjective judgements on the part of the tree improvement specialist. What is likely to be more critical: Survival under drought or maintaining acceptable growth under water limited conditions? If drought survival is most critical, mechanisms related to water uptake such as increased allocation to roots may be more relevant than WUE and D. In any event, analysis of D should be undertaken in conjunction with other measures of fitness under drought such as biomass allocation to roots, assessment of morphological characteristics (Van Buijtenen et al. 1976), and plant water relations (Major and Johnsen 1996).

Understand phenological patterns if working with a wide range of seed sources. Carbon isotope discrimination integrates ci/ca over the time the tissue was formed. If phenology patterns vary widely among genotypes, they may be responding to different environmental signals. This phenology effect may confound the interpretation the 13C signals. While differences in phenology may be difficult to control, efforts should be made to document key phenological events (date of budbreak, shoot and needle extension patterns, date of bud set) in order to aid in interpreting d13C data.

Develop standardized sample protocols. A growing body of evidence has shown that both branch length and crown position can greatly influence d13C values. Tree improvement specialists designing trials based on isotope analysis must consider these factors. Sampling protocols should be designed to sample branches of similar lengths at similar crown position to ensure that the d13C signal represents differences in WUEi rather than the isotopic signal of the source air.

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