Tree Physiology 23, 553–559
© 2003 Heron Publishing—Victoria, Canada
Leaf chlorophyll, net gas exchange and chloroplast ultrastructure in
citrus leaves of different nitrogen status
BHASKAR R. BONDADA1–3 and JAMES P. SYVERTSEN1
1
University of Florida, Citrus Research and Education Center, 700 Experiment Station Road, Lake Alfred, FL 33850, USA
2
Present address: University of California, Department of Viticulture and Enology, One Shields Avenue, Davis, CA 95616, USA
3
Author to whom correspondence should be addressed ([email protected])
Received July 22, 2002; accepted October 26, 2002; published online May 1, 2003
Summary One-year-old ‘Cleopatra mandarin’ (Citrus reticulata Blanco) seedlings were raised in a greenhouse and fertilized with nitrogen (N) at four application frequencies. Nitrogen-deficient leaves (86 mmol N m –2 ) had less chlorophyll per
unit area, but a greater chlorophyll a:b ratio than N-fertilized
leaves (> 187 mmol N m –2 ). Leaf dry mass per area (DM
area –1) and total chlorophyll concentration increased linearly
with increasing leaf N, whereas chlorophyll a:b ratio declined.
Net assimilation of CO2 (A CO 2 ) and leaf water-use efficiency
(WUE) reached maximum values in leaves with ~187 mmol N
m –2. Nitrogen-deficient leaves exhibited small chloroplasts
with no starch granules; grana and stroma lamellae that coincided with the accretion of numerous large plastoglobuli in the
stroma disintegrated. High-N leaves had large chloroplasts
with well-developed grana, stroma lamellae and starch granules that enlarged with increasing N concentration. The lack of
an increase in A CO 2 capacity at leaf N concentrations above
187 mmol N m –2 appeared to be correlated with the presence of
numerous large starch granules.
Keywords: chlorophyll a:b ratio, Citrus reticulata Blanco,
grana, photosynthesis, plastoglobuli, starch, thylakoid.
Introduction
As much as 75% of the total nitrogen (N) in a plant is required
for normal chloroplast formation (Hak et al. 1993, Kutik et al.
1995) and synthesis of components of the photosynthetic apparatus including thylakoid membranes and photosynthetic
enzymes (Evans 1989). A low N supply can cause ultrastructural changes brought about by an accumulation of starch
granules in chloroplasts (Kutik et al. 1995). Starch granules increased in size in N-deficient citrus leaves as well as in leaves
of girdled and defruited branches (Schaffer et al. 1986). In Ndeficient bean (Phaseolus vulgaris L.) leaves, the accumulation of starch granules was accompanied by the deformation
and destruction of grana and thylakoids (Carmi and Shomer
1979). Nitrogen is a fundamental constituent of the photosynthetic apparatus because chlorophyll concentration, photo-
synthesis and growth all decrease with N deficiency (Kutik et
al. 1995).
Within a citrus tree canopy, leaf N concentration varies
widely over time and space (Koo and Sites 1956). Although
citrus leaves can accumulate high concentrations of N, net
CO2 assimilation ( A CO 2 ) does not increase at N concentrations
above 200 mmol m –2 and may decrease (Syvertsen 1984,
Lea-Cox and Syvertsen 1996, Romero-Aranda and Syvertsen
1996). Previous studies have not examined effects of high N
on leaf ultrastructure, which might account for the apparent inhibition of photosynthesis at high N concentrations. Because
the ultrastructure of chloroplasts exerts a strong influence on
the biochemical and biophysical properties of thylakoids,
ultrastructural data could elucidate physiological changes
with leaf N concentrations. For instance, chlorophyll (Chl) a:b
ratio and electron transport activity correlate with the ratio of
appressed to non-appressed thylakoid membranes and the
number of thylakoids per granum (Anderson et al. 1973,
Terashima and Evans 1988, Bjorkman and Demmig-Adams
1995). On the other hand, an accumulation of starch grains can
disrupt chloroplast structure and function by interfering with
thylakoid membrane assembly (Pritchard et al. 1997). We hypothesized that the inhibition of photosynthetic activity of
leaves with leaf N concentration > 200 mmol m –2 is related to
changes in the ultrastructure of their chloroplasts. The objective of this study was to investigate changes in leaf gas exchange and chlorophyll concentration in relation to chloroplast ultrastructure and N supply.
Materials and methods
Plant material
One-year-old ‘Cleopatra mandarin’ (Citrus reticulata Blanco)
seedlings with uniform leaf area and N content were grown in
large pots (to avoid root restriction) containing native Candler
sand in an unshaded greenhouse with maximum daily photosynthetic photon flux (PPF) of 1200 µmol m –2 s –1. Maximum/
minimum temperatures in the greenhouse were 33/23 °C and
relative humidity ranged from 30 to 100% during the course of
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BONDADA AND SYVERTSEN
the study. Seedling leaf age was monitored by tagging leaves
with jeweler’s tags at the time of leaf emergence. After
3 weeks, leaves began to yellow as plants continued to grow.
Twenty seedlings were divided randomly into four fertilization regimes with five replicate plants. About 3.2 g of an
N,P,K fertilizer (20:20:20) in 250 ml of water was supplied at
each fertilization. This volume was sufficient to leach some
fertilizer solution to avoid the accumulation of nutrients. The
first group was fertilized daily, and as a result, received 16 g N
per week. The second group was fertilized weekly (3.2 g N per
week), the third group biweekly (1.6 g N every two weeks)
and the fourth group was unfertilized (N-deficient). All plants
were well watered between applications of fertilizer solution.
Gas exchange measurement
Net assimilation of CO2 of single leaves was measured with an
LI-6200 portable photosynthesis system (Li-Cor, Lincoln,
NE) equipped with a 250-cm 3 cuvette. Two fully expanded
mature leaves, about 2 months old, from each plant in each
treatment were measured. The PPF within the cuvette was
supplemented during all measurements with a QbeamTM solid
state LED lighting system (Quantum Devices, Barneveld, WI)
(Tennesseny et al. 1994) set at 850 µmol m –2 s –1, which exceeds saturating irradiance for sun-grown citrus leaves
(Syvertsen 1984). Within the measurement cuvette, average
leaf temperature was 30.4 ± 1.1 (SE) °C and leaf-to-air vapor
pressure deficit (VPD) was 18.4 ± 2.7 (SE) kPa. All measurements were made in the morning (0830–0930 h) to avoid
higher afternoon temperatures and VPD. Leaf water-use efficiency (WUE) was calculated as the quotient of A CO 2 /leaf
transpiration.
Determination of chlorophyll concentration
After gas exchange measurements had been made, leaves
from each treatment were harvested and their areas determined. Two 9-mm diameter discs were punched with a cork
borer from the mid-laminar area of each leaf using and chlorophyll extracted from the disks with N,N-dimethylformamide
for at least 72 h in the dark at 4 °C. Chlorophyll a, b and total
chlorophyll concentrations (mmol Chl m –2 leaf surface area)
(Porra et al. 1989) were calculated from chlorophyll absorptance measured with a spectrophotometer (Ultrospec II, LKB
Biochrom, Cambridge, England) at 647 and 664 nm.
Ward 1964) and lead citrate (Reynolds 1963), and examined
with a Philips 201 transmission electron microscope at 60 kV.
Electron micrographs of 15 chloroplasts in one representative section from each of the five replicate seedlings were analyzed with Image-Pro Plus (Media Cybernetics, Silver Spring,
MD) to quantify the number of starch grains, plastoglobuli,
grana, thylakoids per granum and chloroplast profile (visible
cross-sectional areas) per chloroplast. For measurement purposes, we regarded representative grana as having at least
three thylakoids per granum.
Nitrogen analysis
The remaining leaf tissues were oven-dried for at least 48 h at
72 °C and leaf dry mass per area (DM area –1) calculated for
each leaf. Dried leaves were ground to powder and total N
concentrations determined with a carbon/nitrogen elemental
analyzer (Fisons Instruments, Dearborn, MI). All data were
analyzed for significant differences by one-way analysis of
variance and Duncan’s multiple range test at P ≤ 0.05. When
appropriate, relationships among N concentration, net gas exchange measurements and chloroplast characteristics were investigated by regression analyses and t-tests at P ≤ 0.05.
Results
Leaf N concentration
Leaf N concentration per unit area increased with N application frequency up to weekly application; there was no difference in N concentrations between the weekly- and dailyfertilized plants (Table 1). On a leaf dry mass basis, the mean
–1
N concentrations were 12.4, 26.7, 41.1 and 41.5 mg g DM
for
N-deficient, biweekly-, weekly- and daily-fertilized plants,
respectively. Values less than 22 mg N g –1 are considered deficient for bearing trees (Tucker et al. 1995).
Leaf dry mass and chlorophyll per area
A linear relationship existed between leaf N concentration and
leaf DM area –1 (Figure 1). There was no difference in DM
area –1 between N-deficient and biweekly-fertilized leaves nor
between daily- and weekly-fertilized leaves. There was a 16%
increase in DM area –1 of N-fertilized leaves over N-deficient
leaves.
Chlorophyll concentration and composition
Transmission electron microscopy
Leaves sampled for gas exchange and chlorophyll measurements were also observed by electron microscopy. Several
1–2 mm 2 pieces were excised from the mid-laminar region of
both leaves with a razor blade, fixed in 3% glutaraldehyde
overnight, washed with 0.1 M potassium phosphate buffer, pH
7.2 and postfixed in 2% osmium tetroxide overnight. The leaf
samples were dehydrated in an acetone series and embedded
in Spurr’s resin (Spurr 1969). Gold and silver ultrathin
(70–90 nm) transverse sections were cut with a glass knife and
mounted on 200-mesh copper grids. The leaf sections were
then double-stained with 2% uranyl acetate (Stempack and
Total leaf chlorophyll concentration increased linearly with
leaf N concentration (Figure 2A). Chlorophyll a:b ratio, however, decreased with increasing leaf N concentration (Figure 2B).
Gas exchange rates
Net assimilation of CO2 was highest in the biweekly fertilization treatment (Figure 3A). Increasing fertilization frequency
from biweekly (187 mmol N m –1) to weekly (367 mmol N
m–1) and daily (368 mmol N m –1) treatments did not increase
A CO 2 significantly. Compared with the fertilized treatments,
internal CO2 concentration (Ci) was significantly higher in the
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555
Table 1. Leaf nitrogen (N) concentration and ratios of chlorophyll (chl) concentration, grana per chloroplast and thylakoids per granum to N of citrus seedling leaves raised at four N supply rates.
N regime
N (mmol m –2)
–1
N (mg g DM
)
Chl:N
Grana:N
Thylakoids:N
0N
Biweekly N
Weekly N
Daily N
86 a1
187 b
367 c
368 c
12.4 a
26.7 b
41.1 c
41.5 c
0.0008 b
0.0012 a
0.0012 a
0.0015 a
–2
0.062 a
0.045 a
0.057 a
–
0.062 a
0.082 a
0.079 a
1
2
Values within a column followed by the same letter are not significantly different at P ≤ 0.05.
Values could not be determined due to disintegration of the membrane system.
N-deficient plants (Figure 3B), whereas water-use efficiency
was significantly lower (Figure 3C).
Chloroplast ultrastructure
All chloroplasts from mesophyll cells were in close proximity
with the cell wall adjacent to intercellular air spaces (Figure 4). Chloroplasts from N-deficient leaves had significantly
smaller total cross-sectional area than those of N-fertilized
leaves (Table 2). Furthermore, in N-deficient leaves, chloroplast integrity was lost as shown by the disintegration of grana
and stroma lamellae (Figure 4A). There was an increase in
number, size and area of plastoglobuli concomitant with the
breakdown of grana and stroma lamellae (Figure 4A, Table 2).
Leaves fertilized with N had large chloroplasts with well-developed grana and stroma lamellae and only a few small
plastoglobuli (Table 2) scattered in the stroma (Figures 4B–
D). Grana number and thylakoids per granum per chloroplast
profile increased with increasing leaf N concentration (Figure 5).
ures 4B–D). At high leaf N concentrations, starch granules occupied most of the space within the chloroplast, thus reducing
the size of the stroma. Differences pertaining to starch
granules and shape of the chloroplast were observed in chloroplasts of N-fertilized leaves (Figure 4). In leaves with
187 mmol N m –2, the normal shape of the chloroplast was unmodified by the presence of a few starch granules (Figure 4B).
However, the increased number and cross-sectional area of the
starch granules (Table 2) distorted the disk-shaped chloroplasts in leaves with high N concentrations (367 and 368 mmol
m–2). The accumulation of starch grains compressed the
thylakoids such that they were pushed toward the periphery of
the organelle (Figures 4C and 4D).
Starch accumulation
No starch granules were observed in N-deficient leaves (Figure 4A). As the N concentration increased, the cross-sectional
area and number of starch granules increased (Table 2 and Fig-
Figure 1. Relationship between leaf dry mass per area (DM area –1)
and leaf nitrogen (N) concentration (y = 83.76 + 0.11x, r 2 = 0.85).
Each value represents one leaf. Symbols: 䊊 = no N; 䊉 = biweekly N
fertilization; 䊏 = weekly N fertilization; and 䉮 = daily N fertilization.
Figure 2. (A) Relationship between chlorophyll (chl) and nitrogen (N)
concentrations (y = –0.05 + 1.56x, r 2 = 0.87), and (B) Chl a:b ratio
and leaf N concentration (y = 3.15 – 1.64x, r 2 = 0.61). Symbols: 䊊 =
no N; 䊉 = biweekly N fertilization; 䊏 = weekly N fertilization; and
䉮 = daily N fertilization.
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BONDADA AND SYVERTSEN
ditional layers of mesophyll cells or the formation of thick
mesophyll tissues to increase leaf thickness (Nobel et al. 1975,
Lloyd et al. 1992). A negative relationship between N concentration and Chl a:b ratio (Figure 2B) was found in this study,
reflecting decreased attenuation of light because of increased
lamina thickness; therefore, the accretion of leaf dry mass
must have resulted from the increase in leaf thickness.
Although high leaf N concentrations result in increases in leaf
thickness, A CO 2 of thick citrus leaves does not necessarily increase (Romero-Aranda et al. 1997) because of decreases in
internal CO2 transfer conductance from the substomatal cavity
to the chloroplast stroma (Syvertsen et al. 1995).
Chlorophyll concentration
Leaf chlorophyll concentration has been shown to increase
linearly with total leaf N concentration from 50 to 200 mmol
m –2 for a number of different species grown under high light
conditions (Evans 1989). The linear increase in chlorophyll
concentration with frequency of N fertilization in our study
implied that N enhanced chlorophyll synthesis, especially Chl
b as indicated by the Chl a:b ratio (Figure 2B). Because chlorophyll is embedded in the thylakoid membrane, increased
chlorophyll synthesis would result in the expansion of
thylakoid membrane assembly mediated by increases in the
number of grana and thylakoids per granum (Table 1). The
preferential allocation of N into chlorophyll synthesis with increasing N fertilization by citrus leaves may reflect a lower N
demand in other components of the photosynthetic apparatus.
Chl a:b ratio
Figure 3. Influence of nitrogen (N) concentration on (A) net assimilation of CO2 (A CO 2 ), (B) internal CO2 concentration (Ci ) and (C) leaf
water-use efficiency (WUE). Error bars indicate SE of the mean of 10
replications.
Discussion
Because starch is the major storage carbohydrate in citrus
(Goldschmidt and Golomb 1982, Sanz and Guardiola 1988),
the corresponding increases in leaf DM area –1 (Figure 1) and
starch with leaf N supply (Table 1) imply that starch accumulation contributes to leaf dry mass. Increased dry mass by
starch accumulation is most commonly observed in plants
grown in a CO2-enriched atmosphere (Neales and Nichols
1978, Wulff and Strain 1981). The thylakoid membrane system also increased with leaf N concentration as demonstrated
by the increased number of grana and thylakoids per granum
(Table 1). This expansion of the membrane assembly may
have supplemented increases in leaf dry mass, analogous to increases observed in rice leaves with high N content (Laza et al.
1993). Leaf DM area –1 can be increased by production of ad-
The Chl a:b ratio can be used as an index to characterize the
developmental state of the photosynthetic apparatus (Grover
and Mohanty 1993). The Chl a:b ratio is stable, at about 3, in
fully green leaves of higher plants, but can vary greatly depending on the physiological status of the plant (Schoefs et al.
1998, Kouril et al. 1999). A Chl a:b ratio of approximately 3
was observed in plants fertilized biweekly that had a leaf N
concentration of 187 mmol m –2. This ratio steadily declined
with increasing leaf N concentrations (Figure 2B). The progressive decline in Chl a:b ratio was accompanied by an increase in Chl b synthesis as indicated by increased numbers of
grana and thylakoids per granum (Table 2) and not by a reduction in Chl a. The Chl a:b ratio of citrus leaves was also influenced by ploidy levels. The Chl a:b ratio of thick tetraploid
leaves containing high amounts of N is lower than that of diploid leaves (Romero-Aranda et al. 1997).
Net gas exchange characteristics and chloroplast
ultrastructure
The relationship between A CO 2 and leaf N showed that maximum rates of A CO 2 are reached at moderate leaf N concentrations (Evans 1983, 1989, Romero-Aranda and Syvertsen
1996). The ultrastructure of chloroplasts provides important
information about the biochemical properties of the thylakoids, such as Chl a:b ratio and electron transport activity,
which were correlated with the ratio of appressed to nonappressed thylakoid membranes and the number of thylakoids
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557
Figure 4. Transmission electron micrographs of chloroplasts from leaves of
citrus seedlings treated with (A) no nitrogen (N), showing plastoglobuli (P),
which increased in number and size,
and disintegrated grana and stroma
lamellae (indicated by arrows)
(×20,000); or N-fertilized (B) biweekly
(×20,000); (C) weekly (×10,000); and
(D) daily (×10,000) with well-developed grana (G), stroma lamellae (SL)
and large starch granules (S). The
grana number, thylakoids per granum
and number of starch granules increased with increasing N
concentration.
per granum (Anderson et al. 1973, Terashima and Evans
1988). Hence, detailed examination of chloroplast ultrastructure may provide insights into the relationship between N
and photosynthesis, especially at high N concentrations.
Transmission electron micrographs of chloroplasts from
N-deficient leaves illustrated loss of membrane integrity (Figure 4A). This corresponded with an increase in size and number of plastoglobuli (Figure 4A, Table 2) and thylakoid
membrane assembly breakdown, implying a synchrony between membrane degradation and plastoglobulus formation
(Lichtenthaler 1968). With increases in leaf N concentration,
there was an increase in granum number until the leaves attained a leaf N concentration of about 300 mmol m–2. Granum
number appeared to be unaffected by further increases in leaf
N concentration (Figure 5A). Thylakoids per granum in-
creased linearly with increasing N concentration (Figure 5B),
indicating that additional N was used for stacking thylakoids
into grana as shown by the declining Chl a:b ratio with leaf N
concentration (Figure 2B), the low ratio of granum:N and high
number of thylakoids per granum:N ratio at high N concentrations (367 and 368 mmol m –2, Table 1).
Simpson et al. (1989) reported that stacking of thylakoids
into grana requires a low Chl a:b ratio, which explains the negative relationship between leaf N concentration and Chl a:b ratio (Figure 2B), and thus, the increased thylakoid number per
granum with increasing leaf N concentration. Leaves containing more than 187 mmol N m –2 had more grana and thylakoids
per granum, but A CO 2 was unaffected (Figure 3A). Thus, the
reduced number of granal thylakoids observed in the biweekly
fertilized leaves relative to the most frequently fertilized
Table 2. Characteristics of chloroplast ultrastructure as affected by nitrogen (N) nutrition of citrus leaves. Abbreviations: Chl length = longest dimension of chloroplast; Chl area = area of chloroplast profile; Pg number = number of plastoglobuli per chloroplast; Pg area = area of
plastoglobuli; St number = number of starch grains per chloroplast profile; and St area = area of mesophyll chloroplast starch grain profiles.
N regime
Chl length (µm)
Chl area (µm2)
Pg number
Pg area (µm2)
St number
St area (µm2)
0N
Biweekly N
Weekly N
Daily N
2.8 c1
5.2 b
7.5 a
8.8 a
3.25 c
5.85 b
8.87 a
9.79 a
9.50 a
4.50 b
2.83 c
3.03 c
0.64 a
0.20 b
0.28 c
0.24 c
–2
2.14 c
4.66 b
7.00 a
–2
3.02 b
6.26 a
7.84 a
1
2
Values within a column followed by the same letter are not significantly different at P ≤ 0.05.
Indicates absence of starch grains.
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BONDADA AND SYVERTSEN
N m –2, chloroplast shapes were unaffected (Figure 4B). High
starch accumulation in weekly- and daily-fertilized leaves indicated that surplus N could not mobilize starch out of the
chloroplasts because most of the N was preferentially partitioned into chlorophyll synthesis (Table 1). Large starch granules in chloroplasts may have limited A CO 2 because of end
product inhibition, a phenomenon most commonly found in
plants exposed to elevated atmospheric CO2 concentrations
(Makino and Tadahiko 1999, Sawada et al. 2001). Because
CO2 transfer conductance strongly depends on the chloroplast
surface area adjacent to the plasma membrane (von Caemmerer and Evans 1991), the morphological modification of
chloroplasts stimulated by starch accumulation at high N concentrations (Figure 5C and 5D) may be another important factor in decreasing CO2 transfer conductance and A CO 2 . Hence,
in leaves with excess N, the N-induced starch accumulation
may be a component in the down-regulation of photosynthesis
that sometimes occurs in plants exposed to elevated CO2 concentrations (Ludewig et al. 1998).
Conclusions
Figure 5. Relationships between (A) nitrogen (N) concentration and
granum number (y = –10.54 + 0.15x – 0.000018x 2, r 2 = 0.88) and (B)
N and thylakoids per granum (y = –7.04 + 0.09x, r 2 = 0.96). Each
value represents one leaf. Symbols: 䊊 = no N; 䊉 = biweekly N fertilization; 䊏 = weekly N fertilization; and 䉮= daily N fertilization.
plants was still adequate to achieve maximum A CO 2 .
Starch accumulation
In leaves with sufficient N supply, N can stimulate mobilization of starch out of the chloroplast to sites of high carbon sink
activity, whereas in N-deficient leaves, starch can build up in
chloroplasts (Ariovich and Cresswell 1983). However, no
starch granules were observed in N-deficient leaves (Figure 4A). This was probably because the 2-month-old N-deficient leaves, prior to the 2–3 week N deprivation treatment,
were initially N-sufficient and had well developed membrane
systems, but no starch granules, presumably owing to
translocation to sites of high sink activity. As the leaves became N-deficient, their membrane system disintegrated as a
result of the accumulation of large plastoglobuli in the stroma
(Figure 4A). Therefore, the decrease in A CO 2 in N-deficient
leaves was a direct consequence of the breakdown of their
thylakoid assembly.
The most intriguing feature of N fertilization was that N enrichment greater than 187 mmol m –2 promoted starch accretion in chloroplasts. The starch granules caused swelling and
distention of chloroplasts in high-N (367 and 368 mmol m –2)
leaves (Figures 4C and 4D), whereas in leaves with 187 mmol
There were major ultrastructural changes in citrus leaf
chloroplasts that correlated with chlorophyll concentration
and gas exchange activities. The chloroplasts from N-deficient
leaves were small, starchless and low in chlorophyll concentration. Furthermore, they exhibited a decrease in A CO 2 and
loss of membrane integrity manifested by the disintegration of
the thylakoid membrane assembly, which coincided with the
accretion of large plastoglobuli in the stroma. Nitrogen-fertilized leaves had high A CO 2 and chlorophyll, and displayed
chloroplasts with large grana and stroma lamellae. An interesting feature of N-fertilized leaves was related to the accretion of starch granules, which distended and distorted the
normal disk shape of chloroplasts in leaves with 367 and
368 mmol N m –2, whereas the normal shape was preserved in
chloroplasts of leaves with 187 mmol N mm –2. Although total
chlorophyll concentration, leaf DM area –1 and thylakoids per
granum increased with leaf N concentration, there were no
significant increases in A CO 2 past the leaf N concentration of
187 mmol m –2, most plausibly because of an increase in starch
granules.
Acknowledgments
The authors thank Ms. Diann Achor for her technical assistance with
electron microscopy. This research was partially supported by a grant
from the Florida Citrus Production Research Advisory Council and
the Florida Agricultural Experiment Station. Approved as Journal Series No. R-08750.
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