Tree Physiology 26, 1067–1073
© 2006 Heron Publishing—Victoria, Canada
Carbonic anhydrase in Tectona grandis: kinetics, stability, isozyme
analysis and relationship with photosynthesis
ANITA TIWARI,1 PRAMOD KUMAR,1 PRAVIN H. CHAWHAAN,1 SANJAY SINGH1 and
S. A. ANSARI1,2
1
Genetics and Plant Propagation Division, Tropical Forest Research Institute, P.O.- R.F.R.C., Jabalpur 482 021 (M.P.), India
2
Corresponding author ([email protected])
Received June 20, 2005; accepted November 11, 2005; published online May 1, 2006
Summary Carbonic anhydrase (CA, EC: 4.2.1.1) activity in
teak (Tectona grandis L.f.) was studied to determine its characteristics, kinetics and isozyme patterns. We also investigated
effects of leaf age, plant age and genotype on CA activity and
gas exchange parameters. Carbonic anhydrase extracted from
leaves in 12 mM veronal buffer, pH 7.8, had a Km for CO2 of
15.20 mM and a Vmax of 35,448 U mg – 1 chlorophyll min – 1,
which values declined by 50 and 70%, respectively, after
1 week of storage at 4 °C. A 15% native polyacrylamide gel revealed the absence of CA isozymes in teak, with only a single
CA band of 45 kD molecular mass observed across 10 segregating half-sib families and groups of trees ranging in age from
10 to 25 years. Activity remained stable during the first month
in storage at 0 °C, but gradually declined to 25% of the initial
value after 1 year in storage. During the period of active growth
(February–May), maximal CA activity was observed in fully
expanded and illuminated leaves. Significant variation was observed in CA activity across 10 1-year-old half-sib families and
21 5-year-old half-sib families. There was a positive correlation between CA activity and photosynthetic rate in a population of 10-year-old trees (P < 0.005). Positive correlations between CA activity and photosynthetic rate were found in 10 of
21 5-year-old half-sib families (P < 0.005 to P < 0.05), which
showed greater diversity in CA activity than in photosynthetic
characteristics. Thus, CA may serve as a biochemical marker
for photosynthetic capacity in teak genotypes.
thesis. Plant CA activity is regulated by light and Zn and CO2
concentrations (Reed and Graham 1981). The enzyme represents 1–2% of the total soluble protein in leaves of C3 plants,
second only to Rubisco in concentration (Reed and Graham
1981, Okabe et al. 1984). Between 86 and 95% of the total CA
is found in chloroplasts (Okabe et al. 1984, Tsuzuki et al.
1985), and the remainder is largely or exclusively confined to
the cytosol of mesophyll cells (Burnell and Hatch 1988).
Khan (1994) found a correlation between CA activity and
photosynthetic rate and productivity in mustard, suggesting
that CA could serve as a marker of productivity because the
enzyme helps in carbon sequestration, particularly in woody
perennials. However, detailed information on the characteristics of CA in tropical broadleaf trees is unavailable (Tiwari et
al. 2005). Therefore, we studied CA in teak (Tectona grandis
L.f.), a valuable timber tree of southeastern Asia, which occupies about 31 million hectares of forest and has been introduced to many tropical regions of the world, e.g., the Pacific,
much of Africa, the Caribbean and Central and South America
(Tewari 1992). Our objective was to determine if CA could
serve as a marker of productivity in this species. Specifically,
we aimed to optimize the extraction procedure and standardize the assay parameters for CA, characterize its kinetics, determine its stability in storage, establish its location within the
leaf and assess how it varies in activity in relation to tree age
and genotype and leaf photosynthetic capacity.
Keywords: biochemical marker, carbon sequestration, extraction buffer, half-sib families, Km , population, storage, tree age,
Vmax .
Materials and methods
Introduction
Carbonic anhydrase (CA), which catalyzes the reaction: CO2 +
H2O = HCO3– + H +, is widely distributed among prokaryotes
and eukaryotes. The occurrence of CA in plants was confirmed by Bradfield (1947). Subsequently, CA has been demonstrated in many terrestrial plants as well as in cyanobacteria
and algae from fresh water and marine environments. Graham
and Reed (1971) suggested that CA is essential for photosyn-
CA extraction and assay
The CA extraction procedure of Tsuzuki et al. (1985) was followed with some modifications. Briefly, about 0.5 g of leaf tissue from fully expanded and illuminated leaves from teak
seedlings or trees was ground in 1 ml of extraction buffer in a
chilled mortar with pestle. The homogenate was centrifuged at
4 °C for 15 min at 5000 rpm (2300 × g), and the supernatant assayed for CA activity according to Wilbur and Anderson
(1948) and for protein according to Lowry et al. (1951). Chlorophyll content in fresh leaves was determined as described by
Arnon (1949).
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TIWARI, KUMAR, CHAWHAAN, SINGH AND ANSARI
Selection of buffer system To optimize the extraction of CA
from teak leaves, five buffer systems were tested: (1) 100 mM
Tris-HCl (pH 8.3) containing 1 mM EDTA (Edwards and Mohammed 1973); (2) 50 mM Imidazole-HCl (pH 7.5) containing
1 mM DTT, 0.5 mM EDTA and 0.1% Triton X-100 (Keys and
Parry 1990); (3) 100 mM Bicine (pH 8.2) containing 20 mM
MgCl2 and 1 mM EDTA (Majeu and Coleman 1994); (4)
12 mM veronal (pH 7.8) (Sultemeyer 1997); and (5) 50 mM
Hepes-NaOH (pH 7.5) containing 0.5 mM EDTA, 10 mM DTT
and 10% glycerol (Gillon and Yakir 2000). Subsequently, unless indicated otherwise, 12 mM veronal buffer (pH 7.8) was
used for all enzyme extractions (Sultemeyer 1997).
Assay for CA activity To prepare the CO2 substrate, pure CO2
was bubbled through distilled water for 2 h. The CO2 concentration in the gas-saturated water was measured by back titration against 0.1 N oxalic acid solution after the addition of
0.1 N NaOH to the carbonic acid solution or a CO2-free distilled water control. The titration end point was detected with
bromothymol blue as the indicator.
For the CA assay method of Wilbur and Anderson (1948),
the reaction mixture was prepared in glass tubes in an ice bath
by mixing 20 µl diluted crude enzyme extract with 1 ml of
veronal buffer containing 20 ppm bromothymol blue (pH 8.3)
to which 1 ml of CO2-saturated water was mixed gently and
the time for the blue color to change to a distinct yellow (Tc)
recorded. The non-enzymatic reaction rate was measured by
adding the CO2 saturated water to the buffer without enzyme
(Tb). The assay was repeated five times with the same leaf extract. A CA unit (U) was calculated as 10(Tb /Tc – 1) and activity was expressed as U mg – 1 chlorophyll or protein min – 1.
molecular mass of the samples by reference to the protein
markers. The other half of the gel was stained with bromothymol blue following the procedure of Edwards and Patton
(1966) for the determination of isozyme patterns.
In vitro stability of CA
Fully expanded and illuminated leaves were randomly collected from five 10-year-old trees and leaf extracts were stored
for 1 year at 0 °C for weekly monitoring of CA activity, expressed as U mg – 1 chlorophyll min – 1.
Leaf position for maximum CA activity
Leaves of different ages (positions) representing the immature
top leaves (L0) and the fully expanded mature first (L1), second (L2), third (L3), fourth (L4) and fifth (L5) leaves from
fully illuminated 10-year-old trees were harvested for extraction and determination of CA activity (U mg – 1 chlorophyll
min – 1). Ten assays per leaf age group were made, taking
leaves from different plants each time.
Seasonal variation in CA activity
Seasonal variation in CA activity was estimated in February,
May, August and November 2002, in five randomly collected
leaves from each of five 10-year-old trees in each month. Enzymatic activity was expressed as U mg – 1 chlorophyll min – 1.
CA activity and isozyme patterns in trees of differing ages
and seedlings of 10 genotypes
To determine Km and Vmax, the enzyme was extracted in 12 mM
veronal buffer, pH 7.8. Ten replicates of each extract were assayed at each CO2 concentration. Ten concentrations of CO2
from 4 to 40 mM CO2 in increments of 4 mM CO2 were prepared by diluting CO2-saturated distilled water with distilled
water from which the CO2 had been released by boiling. Carbonic anhydrase activities of extracts stored for 0, 48 h and one
week after extraction were determined at these CO2 concentrations and Km and Vmax values were determined from plots of
1/CA activity against 1/[CO2].
Semi-hardwood shoot cuttings from trees of 10, 12, 16, 19, 22
and 25 years of age were collected in 2002 from field plantations, rooted in a similar environment in pots and maintained
for one year in earthenware pots at the campus of the Tropical
Forest Research Institute (Ansari et al. 2002). Half-sib seeds
from 10 plus trees (i.e., TNT-20, APKEA-25, APNPL-4,
APJNB-1, ANNPL-5, MHAL-9, MHSC-J2, MHEM-R1,
MHAL-7 and APT-6) were collected, germinated and raised in
pots as 1-year-old half-sib seedlings (Table 1). Subsequently,
fully expanded and illuminated leaves were randomly collected from five rooted trees per age group and 10 seedlings of
each half-sib family for estimation of CA activity and isozyme
analysis as described previously.
Isozyme analysis and apparent molecular mass
Relationship of CA activity with photosynthetic rate
Carbonic anhdrase was extracted from leaves at 4 °C in 50 mM
Tris-HCl buffer, pH 8.3, and centrifuged for 30 min at
10,000 rpm (9300 × g) as described by Harvey and Boulter
(1983). Localization of isozyme and determination of molecular mass were made by polyacrylamide gel electrophoresis.
Aliqots of supernatant and proteins of known molecular mass
(Sigma) were applied to 15% native polyacrylamide gels (all
samples including the markers were loaded in duplicate) and
electrophoresis was performed at 4 °C, using a 25 mM Tris192 mM glycine buffer (pH 8.3) and applying a constant 70 V
electric potential for 6 h. The gel was cut in half and one half
was stained with Coomassie brilliant blue to determine the
We examined the relationship between CA activity and photosynthesis in two populations comprising (1) 21 randomly selected 10-year-old trees from a population of mixed genotypes, and (2) nine 5-year-old trees each of 21 half-sib families
maintained in a progeny trial (Table 1). Both parameters were
measured in two randomly selected fully expanded and illuminated leaves per tree. Enzymatic activity was expressed as (1)
U mg – 1 chlorophyll min – 1 or (2) U mg – 1 protein min – 1.
Photosynthetic rate (PN), stomatal conductance (gs ), transpiration rate (E ) and leaf CO2 concentration were measured with
an LI-6200 portable photosynthesis system ( Li-Cor, Lincoln,
NE).
Determination of Km and Vmax
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CARBONIC ANHYDRASE IN TEAK
1069
Table 1. Source of half-sib progenies of the 1-year-old potted seedlings (TFRI, Jabalpur, M.P.) and the 5-year-old field-grown saplings (Lohara,
Chandrapur, M.S.) of teak.
State (Location)
One-year-old half sib seedlings
Five-year-old half-sib saplings
Andhra Pradesh
(12.39–19.50° N; 77.12–84.44° E)
APJNB-1, APKEA-25, APNPL-4, APNPL-5, APT-6
–
Madhya Pradesh
(18.13–26.52° N; 74.00–86.20° E)
–
BBC-1, BBC-38, Cl-6, Cl –8, Cl-25, Cl-27,
Cl-43, Cl-45, Cl-50, Cl-65, CSC-16, PL-45
Maharastra
(15.37–22.00° N; 72.40–80.54° E)
MHAL-9, MHSC-J2, MHEM-R1, MHAL-7
MHEM-R2, MHSC-A1, MHSC-J1, MHSC-J2
Orissa
(17.47–22.00° N; 81.24–87.12° E)
–
OR-79I, ORAN-R5, ORANP-12, ORPUB-3,
ORPUB-24
Tamil Nadu
(8.00–13.31° N; 76.14–80.20° E)
TNT-20
–
Statistical analysis
All experiments had a complete randomized design and effects
were evaluated by analysis of variance, followed, where treatment effects were significant, by Duncan’s multiple range
mean separation test (DMRT).
Results
The initial concentration of CO2 in water was 60 mM, which
remained unchanged after 1 week. Thus, the CO2-saturated
distilled water substrate was prepared weekly. The CA activities of extracts prepared in the different extraction buffers followed the pattern: 12 mM veronal > 50 mM Hepes-NaOH >
50 mM imidazole-HCL > 100 mM Tris-HCl > 100 mM Bicine
(Figure 1). Maximum activity recorded in veronal buffer was
16,413 ± 3,282 U mg – 1 chlorophyll min – 1, which was about
three times higher than the lowest CA activity recorded in
Bicine buffer. Values for Km and Vmax of extracts remained constant for 0–48 h following extraction, but declined to about 50
Figure 1. Effects of extraction buffers on carbonic anhydrase (CA) activity in leaves of teak: (1) 100 mM Tris-HCl (pH 8.3) containing
1 mM EDTA; (2) 50 mM Imidazole-HCl (pH 7.5) containing 1mM
DTT, 0.5 mM EDTA and 0.1% Triton-X-100; (3) 100 mM Bicine (pH
8.2) containing 20 mM MgCl2 and 1 mM EDTA; (4) 12 mM veronal
(pH 7.8); and (5) 50 mM Hepes-NaOH (pH 7.5) containing 0.5 mM
EDTA, 10 mM DTT and 10% glycerol. Values are means and the bars
indicate 1 SD of the mean; P < 0.01; n = 5. Chlorophyll (chl) concentration = 0.97 ± 0.12 mg g – 1 leaf fresh mass.
and 70%, respectively, in 1-week-old extracts (Table 2). Native polyacrylamide gel electrophoresis revealed the existence
of only a single CA isozyme in all trees of all ages (Figure 2a)
and all genotypes (Figure 2b) tested, with a molecular mass of
45 kD (Figure 2c). The CA activity appeared to remain stable
during the initial three weeks in storage at 0 °C, but declined
continuously thereafter. After one year in storage, CA activity
was reduced to about 25% of the initial activity (Figure 3).
The youngest fully expanded leaves (L1) had the highest
CA activity, and activity gradually declined with leaf age to
Table 2. Values of Km and Vmax for carbonic anhydrase (CA) of teak.
Assays were conducted in freshly prepared leaf extracts and in leaf
extracts stored at 0 °C for 48 h or 1 week after preparation.
Sample
Vmax (U mg – 1 chlorophyll min – 1) Km (mM CO2)
Fresh
After 48 h
After 1 week
35,448
33,333
24,360
15.20
16.67
7.23
Figure 2. Carbonic anhydrase (CA) isozyme in leaf extracts of teak
(Tectona grandis): (a) different ages (years) of trees (I-25, II-22,
III-19, IV-16, V-12, VI-10); and (b) 1-year-old seedlings from half-sib
families (I-TNT-20, II-APKEA-25, III-APNPL-4, IV-APJNB-1, VAPNPL-5, VI-MHAL-P9, VII-MHSC-J2, VIII-MHEM-R1, IXMHAL-P7, X-APT-6). (c) Molecular mass of CA determined by reference to molecular marker proteins (20 µl of enzyme extract containing 0.8–1.0 mg soluble protein was loaded in each well).
TREE PHYSIOLOGY ONLINE at http://heronpublishing.com
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TIWARI, KUMAR, CHAWHAAN, SINGH AND ANSARI
Figure 3. Stability of carbonic anhydrase (CA) activity in leaf extracts
of teak. Values are means and the bars indicate 1 SD of the mean; P <
0.01; n = 9. Chlorophyll (chl) concentration = 0.85 ± 0.13 mg g – 1 leaf
fresh mass.
L5. However, the unexpanded leaves (L0) exhibited the lowest
CA activity of the leaves examined (Figure 4). Season of the
year also significantly influenced CA activity. Samples collected toward the end of winter (February) and during the peak
of summer (May) exhibited the highest CA activity followed
by samples collected August < November (Figure 5). There
was no significant variation in CA activity across trees ranging
in age from 10 to 25 years old (results not shown). In contrast,
a significant genotypic variation in CA activity was observed
in 1-year-old seedlings obtained from 10 half-sib families,
which constituted six groups segregating for CA activity:
half-sib seedlings of ANPL-5 had the highest CA activity and
those of APKEA-25 had the lowest CA activity, with a 3-fold
difference between these extremes (Figure 6). Similarly,
field-grown 5-year-old saplings of 21 half-sib families exhibited significant variation in CA activity, which was similar to
the variation in leaf CO2 concentration, but exceeded the variations in photosynthetic and transpirational rates and in stomatal conductance (Table 3).
The relationship between CA activity and photosynthetic
rate was significantly positive (P < 0.05) in a population of
10-year-old trees (Figure 7), but was variable in the 5-year-old
saplings of 21 half-sib families (Table 4). Ten of the 21 half-sib
families showed a significant positive correlation (P < 0.005 to
P < 0.05) between CA activity and photosynthetic rate (Ta-
Figure 4. Effect of leaf age (position) on carbonic anhydrase (CA) activity in leaf extracts of teak. Values are means and the bars indicate
1 SD of the mean; P < 0.01; n = 10. Chlorophyll (chl) concentration =
0.70 ± 0.17 mg g – 1 leaf fresh mass.
Figure 5. Seasonal variation in carbonic anhydrase (CA) activity in
leaf extracts of teak. Values are means and the bars indicate 1 SD of
the mean; P < 0.01; n = 5. Chlorophyll (chl) concentration = 1.15 ±
0.16 mg g – 1 leaf fresh mass.
ble 4). Linear regresion analysis yielded a coefficient of determination (r 2) of 0.41 (Figure 7); although the dependence varied among genotypes ranging from 0.34 in MHSC-A1 to 0.87
in CSC-16 (Table 4).
Discussion
In previous studies, several buffers were used to extract CA,
e.g., Tris-HCl (Edwards and Mohammed 1973); ImidazoleHCl (Keys and Parry 1990); Bicine (Majeu and Coleman
1994); veronal (Sultemeyer 1997); and Hepes-NaOH (Gillon
and Yakir 2000). We found the highest CA activity in leaf extracts prepared in 12 mM veronal buffer, pH 7.8, although high
activity was also obtained by extraction with Hepes-NaOH
buffer at pH 7.5.
The kinetic parameters of CA in teak leaves (Table 2) are
within the range of values reported for C3 plants such as pars-
Figure 6. Variation in carbonic anhydrase (CA) activity in leaf extracts
of 1-year-old seedlings from 10 half-sib families of teak (Tectona
grandis). Values are means and the bars indicate 1 SD of the mean;
P < 0.01; n = 10. The DMRT test significantly categorized six groups:
1 (ANNPL-5) > 2 (APT-6) > 3 MHAPL-7 > 4 (MHPL-9, MHSC-J2)
> 5 (AJNB-1, MHEM-R1) > 6 (TNT-20, APKEA-25, APNPL-4).
Chlorophyll (chl) concentration = 2.86 ± 0.17 mg g – 1 leaf fresh mass.
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CARBONIC ANHYDRASE IN TEAK
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Table 3. Carbonic anhydrase (CA) activity and physiological characteristics of field-grown 5-year-old saplings from half-sib families of teak
(Tectona grandis; soluble protein concentration = 40.71 ± 8.16 mg g – 1 leaf fresh mass). Abbreviations: PN = net photosynthetic rate; E = transpiration rate; and gs = stomatal conductance. Within a column, data followed by the same letter(s) did not differ significantly (DMRT).
Half-sib family
CA activity
(U mg – 1 protein min – 1)
PN
(µmol m – 2 s – 1)
E
(m mol H2O m– 2 s – 1)
gs
(mol m– 2 s – 1)
Leaf CO2
(ppm)
MHSC-A1
MHSC-J1
MHSC-J2
MHEM-R2
PL-45
BBC-1
BBC-38
CSC-16
Cl-6
Cl -8
Cl-25
Cl-27
Cl-43
Cl-45
Cl-65
OR-79I
Cl-50
ORPUB-3
ORPUB-24
ORAN-R5
ORANP-12
1435 abcd
1674 abcd
1719 abcd
1435 abcd
1350 abcd
1330 bcd
1426 abcd
1640 abcd
2131 a
1541 abcd
1906 abc
1339 bcd
1920 abc
1106 d
2035 ab
1479 abcd
1949 abc
1716 abcd
1157 cd
1846 abcd
1110 cd
21.0 b
24.2 ab
23.8 ab
22.3 ab
22.1 ab
21.4 b
23.0 ab
20.4 b
23.6 ab
25.9 ab
23.7 ab
26.1 ab
20.6 b
25.9 ab
21.2 b
20.4 b
22.0 ab
30.0 a
22.4 ab
18.3 b
23.6 ab
0.98 b
1.07 ab
0.99 b
1.07 ab
1.02 ab
0.97 b
0.93 b
1.06 ab
1.04 ab
1.07 ab
1.11 ab
1.13 ab
1.19 ab
1.00 ab
1.06 ab
1.04 ab
1.00 ab
1.12 ab
1.23 ab
1.04 ab
1.36 a
1.04 c
1.27 abc
1.09 bc
1.34 abc
1.33 abc
1.24 abc
1.14 bc
1.35 abc
1.41 abc
1.38 abc
1.46 abc
1.43 abc
1.57 ab
1.36 abc
1.43 abc
1.54 ab
1.67 a
1.69 a
1.23 abc
1.03 c
1.39 abc
328 abcd
338 abcd
338 abcd
361 abc
392 a
336 abcd
378 ab
371 abc
386 a
377 ab
333 abcd
361 abc
336 abcd
349 abcd
314 bcd
356 abc
347 abcd
281 d
306 cd
304 cd
336 abcd
ley (Tobin 1970) and spinach (Pocker and Ng 1973). We observed declines in Km and Vmax values in 1-week-old leaf
extracts that may be associated with conformational changes
in the active site of the CA protein because active sites are usually the most labile region of the enzyme molecule (Shoichet
et al. 1995, Fan et al. 1996, Zang et al. 1997). Recently, Zoldak
et al. (2004) demonstrated alterations in the Km of NADH
oxidase as a result of changes in flexibility of its active site induced by both chaotropic and kosmotropic anions of Hofmeister series. Like NADH oxidase (Zoldak et al. 2004), CA is
also a highly stable monomeric enzyme (Tiwari et al. 2005)
and the veronal buffer used for enzyme extraction contains
Figure 7. Relationship between CA (carbonic anhydrase) activity and
photosynthetic rate in 10-year-old trees from a population of teak.
Chlorophyll (chl) concentration = 0.97 ± 0.21 mg g – 1 leaf fresh mass.
Correlations are significant at P < 0.005 (n = 19).
chaotropic anions (barbiturate), which may explain the observed reduction in kinetic properties of CA in 1-week-old
enzyme extracts (Table 2).
Table 4. Relationship between carbonic anhydrase (CA) activity and
photosynthetic rate in field-grown 5-year-old saplings from half-sib
families of teak. Asterisks indicate significant correlations at: P <
0.005 (****); P < 0.01 (***); P < 0.025 (**); P < 0.01(*); and ns = not
significant (n = 7).
Half-sib family
Coefficients of determination (r 2 )
MHSC-A1
MHSC-J1
MHSC-J2
MHEM-R2
PL-45
BBC-1
BBC-38
CSC-16
Cl-6
Cl-8
Cl-25
Cl-27
Cl-43
Cl-45
Cl-65
OR-79I
Cl-50
ORPUB-3
ORPUB-24
ORAN-R5
ORANP-12
0.342 *
0.216 ns
0.170 ns
0.565 ***
0.503 **
0.019 ns
0.372 *
0.878 ****
0.490 **
0.066 ns
0.025 ns
0.001 ns
0.120 ns
0.022 ns
0.056 ns
0.100 ns
0.354 *
0.486 **
0.170 ns
0.593 ***
0.366 *
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TIWARI, KUMAR, CHAWHAAN, SINGH AND ANSARI
The distribution of CA isozymes across angiosperm species
is variable. For example, among 24 members investigated,
Triticum vulgaris L. (Poaceace), Raphanus sativus L. (Brasicaceae) and Hydrangea macrophylla (Thunb.) Ser. (Saxifragaceae) lack CA isozymes (Atkins et al. 1972), whereas CA
isozymes have been isolated from tomato (Kositsin and Khalidova 1971), pea (Kachru and Anderson 1974) and lettuce
(Walk and Metzner 1975). Lantana camara L., a member of
the same family (Verbenaceae) as teak, exhibits at least two
CA isozymes (Atkins et al. 1972) and has global distributions
as the most invasive weed in 51 countries (Jenkins and Pimm
2003). Native polyacrylamide gel electrophoresis analysis revealed only a single band of the enzyme across the various age
groups (Figure 2a) and segregating diverse half-sib families
(Figure 2b) of teak, indicating the absence of CA isozymes in
this species. This observation implies that a narrow genetic
base is responsible for the natural distribution of teak in India
below 24° N and in other tropical regions of south-east Asia
with similar geo-climatic conditions (Tewari 1992). The
45 kDa molecular mass of teak CA (Figure 2c) is more similar
to that of the CA protein of monocots (Atkins et al. 1972) than
of dicots (Rossi et al. 1969, Tobin 1970, Atkins et al. 1972,
Kisiel and Graf 1972, Pocker and Ng 1973, Atkins 1974, Walk
and Metzner 1975).
To evaluate CA as a biochemical marker for growth and productivity in the field, it is important to determine the most appropriate stage of leaf development and the time of year for
optimum enzyme activity. Our finding that CA activity was
highest in the first fully expanded and illuminated leaf on the
shoot (Figure 4) is consistent with the observation that the 5′
flanking region of the CA gene contains sequences with homology to the G box, GT box and I box (Kim et al. 1994). These
motifs play roles in tissue-specific and light-modulated expression of the small subunits of Rubisco (Green et al. 1987,
Giuliano et al. 1988), indicating that CA synthesis is light dependent similar to that of the photosynthetic apparatus. This
finding is corroborated by the observation that highest CA activity (Figure 5) occurred during the period of active growth of
new shoots and leaves in the early part of the growing season
when irradiance and photoperiod were maximal (Ansari et al.
2001).
Despite the absence of CA isozymes in teak, there was significant variation in CA activity among 1-year-old potted
seedlings from 10 half-sib families (Figure 6) and among
5-year-old field-grown saplings of 21 half-sib families (Table 3), resulting in CA activity falling into six and four groups,
respectively. The 5-year-old saplings of the 21 half-sib families exhibited a similar grouping of CA activities as found for
CA activity in response to leaf CO2 concentrations (Table 3).
In contrast, the half-sib families showed little variation in
photosynthetic parameters, exhibiting only two groups each
for photosynthetic rate and transpiration rate and three for
stomatal conductance (Table 4). There was no significant difference in CA activity across teak trees varying in age from
10 to 25 years (results not shown).
Carbonic anhydrase activity was significantly and positively correlated with photosynthetic rate in a general popula-
tion of 10-year-old trees (P < 0.005), contributing 41% to
photosynthesis (Figure 7). Similarly, 10 out of 21 5-year-old
teak half-sib families showed a significant positive correlation
between CA activity and photosynthetic rate (r 2 = 0.34 to
0.88) with CSC-16 > MHEM-R2 = ORAN-R5 > PL-45 = Cl-6
= ORPUB-3 > MHSC-A1 = BBC-38 = Cl-50 = ORANP-12
(Table 4). These findings are consistent with those for the C3
agricultural crop, Brassica juncea L. (Khan 1994) and the tree
species, Paulownia tomentosa Steud. (Lazova et al. 2004).
Thus, CA activity is a better marker for photosynthetic capacity than gas exchange for genetic diversity analyses as well
as for identification and field selection of distinctive groups of
teak genotypes exhibiting efficient carbon sequestration. Exploitation of such genotypes should help enhance biomass
production of teak on a sustainable basis and thereby help mitigate the ill effects of global warming.
Acknowledgements
The work was funded by a research project grant (Sanction No. 38
(1011)/01/EMR-II) from the Government of India, Council of Scientific and Industrial Research, New Delhi, which is gratefully acknowledged.
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