‫إستخدام النظائر الكربونية كمعيار إنتخابي لتحسين كفاءة اإلستهالك 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
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 δ13C) 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.
Element
Isotope*
Avg. abundance (%)
Hydrogen
1H
99.985
Hydrogen
2H
0.015
Carbon
12C
98.89
Carbon
13C
1.11
Carbon (Radioactive isotope)
14C
Part per Trillion
Oxygen
16O
99.759
Oxygen
17O
0.037
Oxygen
18O
0.205
* 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
2) Agronomic Method: WUE = Plant Productivity/ET
3) 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
water stress
transpiration
rate
humidity
leaf
conductance
photon flux
CO2

ci
ca
Woody Plants: C3
Environmental causes
of d13C variation
Nitrogen
photosynthetic rate
productivity
Growth,
reproductive output
canopy
leaf area
•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.
Natural Abundance Terminology

Isotopic Ratio
R


Abundance of Rare Isotope
Abundance of Common Isotope
13C/12C
(R)
Delta notation
 Rsample

d  
 1  1000
 Rstandard 

Units (‰) Parts per thousand or “per mil”
d13C = (Rsample/Rstd – 1) x 1000
R = molar ratio of heavy / light isotope
(e.g., 13C/12C)
This gives “delta” notation in “per mil” (‰)
Isotopic composition
13
12


C / Csample
13
d C   13 12
 1 x1000

C
/
C
s
tan
dard


V-PDB standard
CO2 in air
C3 plant biomass
Respired CO2
0 ‰ by definition
-8 ‰ (-7 to -15 ‰ )
-24 to -30 ‰
-24 to -30 ‰
per mil
Carbon Isotope Discrimination
a measure of Intrinsic Water-Use Efficiency
 ci 

  a  (b  a )
c 
 a 
∆ = 4.4 + 22.6(Ci /Ca)
WUE
intrinsic

(
C

C
)
A i a
g
16
.
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 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, WUEi).
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).
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.)
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:
1) 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. 1994).
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.
Advantages of D as a selection criteria for
improved WUE
1) Carbon isotope discrimination has several
conceptual and logistical advantages to
screening for drought tolerance based on TE or
WUEi.
2) Carbon isotope discrimination integrates ci/ca
over the time the sampled tissue was formed.
In contrast, WUE measured by gas exchange
provides ‘snapshots’ of A/g or A/E and may not
be representative of overall WUE.
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.
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.
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.
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.
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 phonology
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 phonology
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.
Conclusion
From the foregoing discussion we may conclude the following:
The carbon isotope composition of plant tissue is
1) physiologically linked and correlated with WUE and
TE
2) Varies significantly among genotypes in many crops
and trees
3) stable across years and moisture regimes
4) Can be used to rapidly sample a large number of
genotypes in multiple locations
5) Can be used to sample physiological response to past
environments in trees using increment cores or past
year’s foliage