إستخدام نظائر الكربون كمعيار إنتخابي لتحسين كفاءة الاستهلاك المائي في المحاصيل الزراعية Carbon Isotope Discrimination as a Selection Criterion for Improved Water-Use Efficiency in Agricultural Crops By Dr. Ali Abdullah Alderfasi Professor of Crop Physiology Plant Production Department King Saud University First Semester 1431

Introduction Nutrient and water management practices are the main factors affecting in increasing crop production in arid /semi-arid areas. Carbon isotope discrimination (CID ) has been proposed as physiological criterion for predicting water use efficiency (WUE) in crops and trees. Selection for improved WUE through analysis of carbon isotopes will be most useful in selection for maintenance of growth under drought environments such as Saudi Arabia

The isotopic ratio of 13C to 12C in plants tissue is less than the isotopic ratio of 13C to 12C in the atmosphere, indicating that plants discriminate against 13C during photosynthesis. Such discrimination against 13C (i.e., difference between 13C and 12C, expressed as delta δ 13C) in plant tissues (leaves and grains) has been successfully used in the selection of drought resistant in barley, wheat, rice and peanut and many other crops and trees under water-limited environments. In contrast, for well-water environments , many positive genotypic correlations have been reported between delta and grain yield indicating potential value in selecting for greater delta in these environments.

Water needed for food production(Liters of water per kilogram of food) (http://www.fertilizer.org/ifa/Form/pub_position_papers_8.asp)

Terrestrial abundance of the stable isotopes of some important elements used in ecological studies. Avg. abundance (%) Carbon 12C 98.89 13C 1.11 Carbon (Radioactive isotope) 14C Part per Trillion Hydrogen 1H 99.985 2H 0.015 Oxygen 16O 99.759 17O 0.037 18O 0.204 Nitrogen 14N 99.63 15N 0.37 * Isotopes are atoms with same # protons but different # neutrons.

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

Scientific Bases

WUE in plants can be measured by the following methods:- 1) Physiological Method: WUE = A/T or A/gs Agronomic Method: WUE = Plant Productivity/ET Use of Carbon Isotopic Discrimination (CID) as Indirect Method: D = 4.4 + 22.6(Ci / Ca) Selection for improved WUE through analysis of carbon isotopes will be most useful in selection for maintenance of growth under drought environments

 Woody Plants: C3 water stress transpiration rate humidity leaf conductance photon flux canopy leaf area CO2 Nitrogen ci ca photosynthetic rate  productivity Woody Plants: C3 Environmental causes of d13C variation Growth, reproductive output 12

Variation in discrimination against 13C during photosynthesis is due to both stomata 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. * Moreover, in woody plants, carbon isotope discrimination can be determined on annual ring samples, providing a historical analysis of plant response to environmental conditions

Carbon isotope measurements * Samples are easily collected, and processed and large numbers of samples may be collected in diverse environments. * Samples are harvested and dried at 70 o C for 48 hours and samples are ground to pass a 1-mm sieve. Carbon isotope composition of each sample is determined by mass spectrometry.

      Mass Spectrometer In order to measure the characteristics of individual molecules, a mass spectrometer converts them to ions so that they can be moved about and manipulated by external electric and magnetic fields. The three essential functions of a mass spectrometer, and the associated components, are: 1.   A small sample is ionized, usually to cations by loss of an electron. The Ion Source 2.   The ions are sorted and separated according to their mass and charge.   The Mass Analyzer 3.   The separated ions are then measured, and the results displayed on a chart.   The Detector

Diagram of a simple mass spectrometer The basic components of a mass spectrometry system. Ionization Source Mass Analzyer Detector selected ions Data System Inlet all ions

Fig. 13.39

Natural Abundance Terminology Isotopic Ratio 13C/12C (R) Delta notation (13C ) ,Carbon Isotope Composition Units (‰) Parts per thousand or “per mil”

13C(‰) = (R sample /R std – 1) x 1000 R = molar ratio of heavy / light isotope (e.g., 13C/12C) Carbon isotope composition 13C or d13C converted to carbon isotope discrimination ∆ or D using the following equation (Farquhar et al., 1989) ∆ (‰) = (13Ca - 13Cp)/ (1+ 13Cp) The standard is the CO2 obtained from limestone from Pee Dee Belmenite (PDB) formation in south Carolina, USA (0 ‰ by definition)

Carbon Isotope Discrimination a measure of Intrinsic Water-Use Efficiency ∆ = 4.4 + 22.6(Ci /Ca) Where a = discrimination against 13C due to diffusion through stomata (4.4‰) , b = discrimination against 13C due to carboxylation (27‰), ci internal [CO2], ca ambient [CO2]

* 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 (ci ) 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).

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, WUE ).

The amount of isotopic discrimination that occurs during assimilation may be compared by D or d13C(Carbon isotope composition) . 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).

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 wheat grass 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 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). 2) wheat (Triticum aestivum L.) (Farquhar and Richards 1984 and Condon et al. 1990).

3) peanut (Arachis hypogea L. ) (Hubick et al. 1986 and Wright et al 4) barley (Hordeum vulgare L.) (Acevedo 1993), 5) cowpea (Vigna unguiculata [L.] Walp.) (Ismail et al. 1994).

Figure 1. Relationship between 13C discrimination of seeds and WUE barley under water stress.

Figure 2. Relationship between 13C discrimination of leaves and WUE barley under water stress.

Figure 3. Relationship between 13C discrimination of seeds and aerial dry matter of barley under water stress.

Figure 4. Relationship between 13C discrimination of seeds and grain yield of 6-row barley under water stress.

These studies suggest that genetic variation in D may be sufficient to be useful as a selection criterion for improved water use efficiency in Agricultural crops.

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 WUE 2) Carbon isotope discrimination integrates ci/ca over the time the sampled tissue was formed.

3) 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. In contrast, WUEi measured by gas exchange provides ‘snapshots’ of A/g or A/E and may not be representative of overall WUE.

4) 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)

5) 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).

6) D may be also correlated with productivity 6) 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.

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. 1) Location 2) Plant Height 3) Plant Canopy 4) Branch length 5) Plant phenology 6) Hydraulic Conductivity 7) Cost

Plant Height 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.

Plant Canopy Re-fixing of respired carbon can affect the carbon isotope signal of under story 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

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. Isotope discrimination is related to hydraulic conductivity because stomata close in response to increasing tension in the xylem (Irvine et al. 1999).

Importance of phenology plant phonology 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 phonology.

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. However, these factors make isotope analysis attractive to those interested in selecting genotypes for improved stress tolerance under arid zones. The advantages must be balanced against some potential limitations.

Conclusion From the foregoing discussion we may conclude the following. The carbon isotope composition of plant tissue:- Is physiologically linked and correlated with WUE and TE Varies significantly among genotypes in many crops and trees Is stable across years and moisture regimes Can be used to rapidly sample a large number of genotypes in multiple locations Can be used to sample physiological response to past environments in trees using increment cores or past year’s foliage