Tree Physiology 24, 347–353
© 2004 Heron Publishing—Victoria, Canada
Variations in dark respiration and mitochondrial numbers within
needles of Pinus radiata grown in ambient or elevated CO2 partial
pressure
KEVIN L. GRIFFIN,1,2 O. ROGER ANDERSON,1 DAVID T. TISSUE,3 MATTHEW H.
TURNBULL4 and DAVID WHITEHEAD5
1
Lamont-Doherty Earth Observatory of Columbia University, Palisades, NY 10964, USA
2
Corresponding author ([email protected])
3
Department of Biology, Texas Tech University, Lubbock, TX 79409, USA
4
Department of Plant and Microbial Sciences, University of Canterbury, Christchurch, New Zealand
5
Landcare Research, P.O. Box 69, Lincoln 8152, New Zealand
Received April 3, 2003; accepted September 5, 2003; published online January 2, 2004
Summary Within-leaf variations in cell size, mitochondrial
numbers and dark respiration rates were compared in the most
recently expanded tip, the mid-section and base of needles of
Pinus radiata D. Don trees grown for 4 years in open-top
chambers at ambient (36 Pa) or elevated (65 Pa) carbon dioxide
partial pressure ( p(CO2)a). Mitochondrial numbers and respiratory activity varied along the length of the needle, with the
highest number of mitochondria per unit cytoplasm and the
highest rate of respiration per unit leaf area at the base of the
needle. Regardless of the location of the cells (tip, middle or
basal sections), needles collected from trees grown in elevated
p(CO2)a had nearly twice the number of mitochondria per unit
cytoplasm as those grown in ambient p(CO2)a. This stimulation
of mitochondrial density by growth at elevated p(CO2)a was
greater at the tip of the needle (2.7 times more mitochondria
than in needles grown in ambient CO2) than at the base of the
needle (1.7 times). The mean size of individual mitochondria
was unaffected either by growth at elevated p(CO2)a or by position along the needle. Tree growth at elevated p(CO2)a had a
variable effect on respiration per unit leaf area, significantly increasing respiration in the tip of the needles (+25%) and decreasing respiration at the mid-section and base of the needles
(–14% and –25%, respectively). Although a simple relationship between respiration per unit leaf area and mitochondrial
number per unit cytoplasm was found within each CO2 treatment, the variable effect of growth at elevated p(CO2)a on respiration along the length of the needles indicates that a more
complex relationship must determine the association between
structure and function in these needles.
Keywords: peroxisomes, transfusion tissue, ultrastructure.
Introduction
Although not often studied, there is significant within-leaf
variation in physiological activity (Ma and Dwyer 1997, Mott
and Buckley 2000, Zangerl et al. 2002). If there is significant
within-leaf variation in respiratory activity or associated structural features, it could be important to leaf-level carbon exchange in the context of efforts to quantify and understand
plant responses to global environmental change, particularly
variations in the partial pressure of atmospheric carbon dioxide ( p(CO2)a). A significant effort has been made to better understand, quantify and predict the effects of plant growth at
elevated p(CO2)a on physiological processes regulating the exchange of carbon between the atmosphere and the biosphere
(Griffin and Seemann 1996, Drake et al. 1997, Norby et al.
1999). Information ranging from the ecosystem to molecular
scales exists, yet little is known at the scale of cellular ultrastructure. In particular, we have limited knowledge about the
effects of growth at elevated p(CO2)a on mitochondria (but see
Robertson et al. 1995, Griffin et al. 2001a).
Regulation of the number of mitochondria in cells is not
well understood (Logan and Leaver 2000), but may depend on
cell type (Bowsher and Tobin 2001) and developmental stage
(Thompson et al. 1998). Although the number of mitochondria
varies from hundreds to thousands (Buchanan et al. 2000), it
has often been assumed that the number of mitochondria per
unit cytoplasmic volume is invariant (Buchanan et al. 2000).
However, when comparing plants grown in elevated versus
ambient p(CO2)a, Robertson et al. (1995) reported a threefold
increase in the number of mitochondria in developing leaf
cells grown in elevated p(CO2)a just 12 h post-mitosis in
7-day-old Triticum aestivum L. plants. To assess the generality
of this response, we previously surveyed eight species (representing seven plant families, including both angiosperm and
conifer species) grown in several different growth facilities
with different CO2-dosing technologies. We found that growth
at elevated p(CO2)a increased the number of mitochondria per
unit cell area by 1.3 to 2.4 times compared with the number in
348
GRIFFIN, ANDERSON, TISSUE, TURNBULL AND WHITEHEAD
control plants grown in lower p(CO2)a (Griffin et al. 2001a).
Clearly, plant growth at elevated p(CO2)a can have important
effects on the fine structure of leaf cells, but the link to physiological function is less clear.
Mitochondria contain respiratory machinery and are a primary site of ATP, organic acid and amino acid production via
the citric acid cycle and associated electron transfer chain.
Hence, we may expect that changes in the number or structure
of this essential organelle have important related implications
for cellular function. In our previous study (Griffin et al.
2001a), we found an increase in mitochondrial number in
leaves of species grown in elevated p(CO2)a, but a decrease in
the rate of mass-based dark respiration. Although both shortand long-term respiratory responses to elevated CO2 partial
pressure have been demonstrated (Poorter et al. 1992, Gonzalez-Meler et al. 1996, Curtis and Wang 1998, Drake et al.
1999), the precise mechanisms have not been identified, and
the magnitude and even the direction of leaf-level responses
are quite variable. Therefore, given the uncertainty in the
mechanisms regulating leaf-level respiration, we are unable to
predict the response of respiration at the ecosystem scale to
global environmental change.
There is a paucity of information on the functional anatomy
of conifer needles, especially physiological correlates of fine
structure at different stages of needle development. Yet
Bowsher and Tobin (2001) argue that developmental changes
within plant tissues offer a practical system to study the relationship between mitochondrial form and function. In this
study we used such a system, measuring variation in cell size,
mitochondrial numbers and dark respiration rates within different aged portions of Pinus radiata D. Don needles (the most
recently expanded tip, the mid-section and the base of the needle) from trees grown in ambient or elevated p(CO2)a. Pine
needles elongate from the tip over a period of several weeks to
months, creating a gradient in cell ages and, therefore, in fine
structure and physiological function (Esau 1977). Previously,
we reported that the mid-section of P. radiata needles responded similarly to that of all other species examined, with
2.1 times more mitochondria and 14% lower rates of respiration per unit leaf area when trees were grown in elevated
p(CO2)a compared with trees grown in ambient p(CO2)a (Griffin et al. 2001a). Here, we present new data collected from the
tip and basal sections of the same needles with the aim of elucidating how variations in p(CO2)a affect cellular fine structure
and respiratory activity during needle development. The axial
tissue gradient in developing pine needles provides an excellent system to examine possible interactive effects of variations in p(CO2)a on foliage structure and physiology during
needle ontogeny. Considering structure and function independently, we hypothesized that the largest number of mitochondria per unit area of cytoplasm, the lowest respiration
rates per unit leaf area (i.e., lowest metabolic activity) and the
least sensitivity to growth in elevated p(CO2)a (i.e., because of
lower rates of respiration and photosynthesis) would occur at
the base of the needle (i.e., most mature cells).
Materials and methods
Growing conditions
Our experimental site was established in 1994 at Christchurch,
New Zealand (43°32′ S, 172°42′ E, elevation 9 m a.s.l.). Sixteen circular open-top chambers (3.6 m tall, 4.6 m diameter,
see Heagle et al. (1989)) were established on a recently stabilized free-draining dune sand. The design and performance of
the chambers are described elsewhere (Whitehead et al. 1995).
The CO2 supplied to the chambers was obtained by separation
from biogas using a three-stage filtration process at a nearby
wastewater treatment facility (Rogers and Whitehead 1998).
Experimental p(CO2)a treatments were monitored and controlled automatically, and applied continuously for the entire
life of the trees. Three Pinus radiata trees were planted in each
half chamber. All experimental measurements were made on
trees grown from clonal, tissue-cultured stock at ambient
(36.2 ± 0.37 Pa) or elevated (65.4 ± 0.69 Pa) p(CO2)a. These
clones were propagated from the bud of a 4-year-old tree. During the tissue culture and subsequent rooting processes, the
trees were maintained in the appropriate ambient and elevated
p(CO2)a treatments. The physiological, biochemical and morphological measurements presented here were made during
the early growing season (November 1996) when the trees
were 4 years old and 2–3 m high. The full complement of biochemical, physiological and morphological measurements,
which was made on fully exposed, north-facing branches using fully expanded needles produced the previous spring
(“1-year-old needles,” age 14 months), has been described previously by Griffin et al. (2000). Needles from two fascicles
were measured from the appropriate cohort on secondary
branches of single trees from six chambers per CO2 treatment.
Respiration measurements
Needles were sampled at the tip (most distal 20 mm of the needle from the fascicle), the mid-section (central 20 mm segment) and the base (20 mm directly above the fascicle sheath).
Respiration was measured on segments of needles prior to fixation for transmission electron microscopy. Needle segments,
about 5 mm long, were placed in the cuvette and depletion of
oxygen was assayed with a Clark-type liquid-phase oxygen
electrode (Rank Brothers, Cambridge, U.K.) in the dark at
25 °C in 20 mM MES buffer (pH 6.0) that had been equilibrated in ambient air (Azcón-Bieto et al. 1994).
Measurement of the numbers and size of mitochondria
Samples from the same needles as those used for the respiration measurements were cut into 1-mm slices and fixed immediately in 2% glutaraldehyde (w/v) in 0.05 M potassium
phosphate buffer, pH 7.2. Samples were post-fixed in phosphate-buffered 2% osmium tetroxide (w/v) for 2 h at 5 °C, dehydrated in a graded acetone series, embedded in catalyzed
epon (TAAB resin, Energy Beam Sciences, Agawam, MA),
polymerized at 75 °C, and sectioned with a Porter-Blum MT-2
ultramicrotome fitted with a diamond knife. Sections were
collected on copper grids, post-stained with Reynolds lead ci-
TREE PHYSIOLOGY VOLUME 24, 2004
FUNCTIONAL ANATOMY AND PHYSIOLOGY OF NEEDLE ONTOGENY
trate and observed with the aid of a Philips 201 transmission
electron microscope (Eindhoven, the Netherlands) operated at
60 kV.
To obtain an estimate of the number of mitochondria per
unit area of cytoplasm lining the cell walls in the images of the
relatively polygonal Pinus cells, we made measurements directly on TEM negatives of cell images recorded at 1000×. We
counted the number of mitochondria within a rectangular area
enclosing the layer of cytoplasm lining the cell wall (length
times thickness of the enclosed layer of cytoplasm expressed
in mm). The area of the cytoplasm was measured with a magnifying reticle with a 0.1-mm scale. The mean density of mitochondria (number per µm2) was calculated by converting the
millimeter measurements to micrometer units, based on the
magnification of the TEM images. At least 50 measurements
of cytoplasmic area (using 50 negatives) were made for each
of the six treatments for a total of 300 measurements. Measurement error was calculated by making two separate measurements of the number of mitochondria per unit area in the
same set of TEM negatives (but in a different sequence each
time to ensure greater objectivity) for a total of 50 measurements. The percent error (difference between replicate means
divided by the grand mean multiplied by 100) was 3.5%.
The length (major axis) and width (minor axis) of individual
mitochondria were also measured directly on the negatives of
electron micrographs with a magnifying reticle with a 0.1-mm
scale. This was done to determine if the sizes of the mitochondria in each segment of the pine needle were similar. At least
30 mitochondria were measured in each of the six treatment
samples, giving a total of 180 measurements.
Mean cell size was also measured to determine if there were
differences among the three segments of the pine needle. Cell
sizes were measured directly on the transmission electron microscope screen at a magnification of 1500×. The measurements were made to a precision of 1 mm on the screen grid
(equivalent to 0.7 µm actual dimension of the cell when adjusted for magnification). Thirty-five to 55 cells were measured for length and width (major and minor axes) for each of
the six treatments. In each of the above measurements, representative chlorophyllous cells for the six treatments were examined in ultrathin sections obtained from the periphery of the
P. radiata needle in a region immediately beneath the epidermis.
Statistical analyses
Respiration measurements were subjected to analysis of variance (ANOVA) using SPSS (Version 10.0.2, SPSS, Chicago,
IL). Comparisons among means for CO2 treatments were
made by least-significant difference (LSD). For mitochondrial
numbers, one-tailed t-tests of mean differences were performed with a STATVIEW SE + graphics computer application (Abacus Concepts, Berkeley, CA).
Results
Tree growth in elevated p(CO2)a had no effect on the timing of
needle growth or on final needle length, but it had a substantial
349
effect on within-leaf cell fine structure. Cells in the apical tissue of needles tended to be smaller with a thicker layer of cytoplasm and relatively smaller vacuole (Figure 1), whereas cells
in the mid-region and base of needles were larger with a thin
layer of cytoplasm and increasingly larger vacuoles. Transfusion, or laterally conducting, tissue was commonly found in
the base of the needle, so care was taken to sample only representative chlorophyllous cells. Tips of needles tended to have
half the number of mitochondria per unit area found in midsections of the same needles (Table 1). The basal sections of
needles, with large vacuoles and a small amount of cytoplasm,
had the highest number of mitochondria per unit area, with
more than 7.5 times the number found in the tips of needles of
trees grown in ambient p(CO2)a, and 4.5 times the number in
the tips of needles of trees grown in elevated p(CO2)a. Growth
in elevated p(CO2)a resulted in a significant increase in the
number of mitochondria per unit area of living cytoplasm at all
three positions in the needles (tip, mid-section and base). We
note that the mitochondrial counts from the mid-section of
these needles were previously reported in the general survey of
mitochondrial responses from a variety of plant species (Griffin et al. 2001a). The stimulation of mitochondrial numbers
per unit area by growth in elevated p(CO2)a was 59% higher at
the tip of the needle (2.7 times) than at the base of the needle
(1.7 times) with an overall mean increase of 1.9 times.
The mean size of individual mitochondria was unaffected
by either growth in elevated p(CO2)a or needle position (Table 2). The shape of individual mitochondria and the general
cellular appearance were also unaffected. On average, the
length of the major axis of the cross section of individual mitochondria was 0.75 µm, and the length of the minor axis was
0.49 µm. Individual cell sizes increased significantly from the
tip to the base of the needle, but were not influenced by the
CO2 treatment (Table 3). Mean cell sizes were 23.8 µm for the
major axis and 15.9 µm for the minor axis, and were about
35% larger at the base than at the tip. Peroxisomes were
counted in the needle tips, and no significant effect of CO2
treatment was found. For trees grown in ambient p(CO2)a, the
mean number of peroxisomes per unit area of living cytoplasm
was 1.3 ± 0.7 µm –2, whereas for trees grown in elevated
p(CO2)a, the mean number of peroxisomes per unit area of living cytoplasm was 1.9 ± 0.6 µm –2 (P > 0.05). The ratio of peroxisomes to mitochondria was greatly reduced, from 0.19 to
0.10, by growth in elevated p(CO2)a.
The rate of respiration was lowest in the tip of the needles
and highest at the base (Table 4). The effect of elevated
p(CO2)a on respiration was variable, increasing respiration significantly at the tip of the needles, and decreasing it at the
mid-section and base of the needles. As a result, the difference
in respiration between the tip and base of the needles was
larger for trees growing in the ambient treatment (220% increase) compared with trees growing in the elevated treatment
(93%). Mean rates of respiration were reduced by 12%, from
0.43 ± 0.02 µmol m –2 s –1 for trees growing in the ambient
treatment to 0.38 ± 0.02 µmol m –2 s –1 for trees growing in the
elevated treatment. As with the mitochondrial counts, respiration rates from the mid-section of the needles have been re-
TREE PHYSIOLOGY ONLINE at http://heronpublishing.com
350
GRIFFIN, ANDERSON, TISSUE, TURNBULL AND WHITEHEAD
Figure 1. Transmission electron micrographs of ultra-thin
sections of Pinus radiata needles taken from the tip (A and
B, the most distal 20-mm of
the needle from the fascicle),
middle (C and D, the central
20 mm segment) and base (E
and F, 20 mm directly above
the fascicle sheath). Samples
shown are from the ambient
CO2 (A, C, and E) and elevated CO2 (B, D, and F) treatments. The cells in the apical
tissue tended to be smaller
with a thicker layer of cytoplasm and relatively smaller
vacuole (V). Cells in the
mid-region and base were
larger with a thin layer of cytoplasm and increasingly larger
vacuoles (e.g., E). Mitochondria (arrows) were more abundant in needles from trees
grown in elevated CO2 partial
pressure compared with those
grown in ambient CO2 partial
pressure. Bar = 5 µm.
ported previously (Griffin et al. 2001a).
Discussion
We found clear differentiation of cellular fine structure and respiratory activity between the newly produced tip and older
base of needles of P. radiata. In addition, both structure and
respiration varied with growth p(CO2)a, and there was a strong
interaction between p(CO2)a and needle position. It has been
recognized that direct physiological responses to plant growth
at elevated p(CO2)a likely have important structural analogs at
multiple spatial scales (Pritchard et al. 1999). However, to our
Table 1. Numbers of mitochondria per unit area of cytoplasm in needles collected from Pinus radiata trees grown in ambient (36 Pa) or elevated
(65 Pa) CO2 partial pressure. Needles were sampled at the tip (most distal 20 mm of the needle from the fascicle) the middle (central 20-mm segment) and the base (20 mm directly above the fascicle sheath).
Sample location
Number of mitochondria per 100 µm2 of cytoplasm
P
Ambient (A) (mean ± SEM)
Elevated (E) (mean ± SEM)
Ratio (E/A)
Tip
Middle
Base
7.0 ± 1.0
14.0 ± 1.4
53.1 ± 7.8
19.0 ± 2.0
30.0 ± 1.8
88.1 ± 11.5
2.7
2.1
1.7
Mean
24.7
45.7
1.9
TREE PHYSIOLOGY VOLUME 24, 2004
< 0.001
< 0.001
< 0.010
FUNCTIONAL ANATOMY AND PHYSIOLOGY OF NEEDLE ONTOGENY
351
Table 2. Length (major axis) and width (minor axis) of mitochondria in cells from the tip (most distal 20 mm of the needle from the fascicle), middle (central 20-mm segment) and base (20 mm directly above the fascicle sheath) of needles of Pinus radiata trees grown in ambient (36 Pa) or elevated (65 Pa) CO2 partial pressure. Trees grown in ambient and elevated CO2 partial pressure were not significantly different based on unpaired
t-tests (df = 58).
Sample location
Mitochondria dimensions (µm)
Length
Width
Ambient (mean ± SEM)
Elevated (mean ± SEM)
Ambient (mean ± SEM)
Elevated (mean ± SEM)
Tip
Middle
Base
0.79 ± 0.21
0.75 ± 0.26
0.70 ± 0.11
0.80 ± 0.24
0.81 ± 0.27
0.66 ± 0.06
0.45 ± 0.08
0.50 ± 0.11
0.51 ± 0.06
0.45 ± 0.10
0.50 ± 0.10
0.53 ± 0.03
Mean
0.75
0.76
0.49
0.49
Table 3. Length (major axis) and width (minor axis) of cells from the tip (most distal 20 mm of the needle from the fascicle), middle (central
20-mm segment) and base (20 mm directly above the fascicle sheath) of needles of Pinus radiata trees grown in ambient (36 Pa) or elevated
(65 Pa) CO2 partial pressure. Cells from trees grown in ambient and elevated CO2 partial pressure were not significantly different based on unpaired t-tests (df = 58).
Sample location
Cell dimensions (µm)
Length
Width
Ambient (mean ± SEM)
Elevated (mean ± SEM)
Ambient (mean ± SEM)
Elevated (mean ± SEM)
Tip
Middle
Base
20.3 ± 0.82
23.0 ± 1.30
29.0 ± 2.37
19.0 ± 1.02
21.0 ± 1.10
30.3 ± 1.29
14.3 ± 0.46
16.0 ± 0.94
18.4 ± 1.23
15.0 ± 0.60
15.5 ± 0.61
16.2 ± 0.87
Mean
24.1
23.4
16.2
15.6
knowledge, variation within needles in ultrastructure and respiration in response to p(CO2)a has not been reported previously.
Gymnosperm needles are differentiated into vascular tissue
containing xylem and phloem, photosynthetic mesophyll
cells, a hypodermis and an epidermis. In addition to these major cell types, a layer of transfusion tissue surrounds the vascular tissue and is enclosed by an endodermis. In the sections
examined, the basal portions of the needles were composed
primarily of transfusion tissue. These cells tended to be large,
with large vacuoles and a relatively small amount of cytoplasm. As a result, there were relatively few mitochondria per
unit total cell area. The morphology and abundance of the
transfusion tissue suggest that the primary function of the base
of the needle is the lateral transport of material between the
vasculature and other parts of the needle (Esau 1977). For the
comparison of mitochondrial numbers, only chlorophyllous
cells obtained from the periphery of the P. radiata needle in a
region immediately beneath the epidermis were examined.
Despite relatively few mitochondria per unit total cell area,
cells from the basal sections of the needles had the highest
numbers of mitochondria per unit area of living cytoplasm.
However, as shown in Figures 1E and 1F, the cytoplasm of the
basal cells is quite thin compared with the more apical portions
of the needle, so the density per unit area of cytoplasm must be
interpreted with this fine structural feature in mind. In con-
Table 4. Rates of respiration in needles from Pinus radiata trees grown in ambient (36 Pa) or elevated (65 Pa) CO2 partial pressure. Needles were
sampled at the tip (most distal 20 mm of the needle from the fascicle) the middle (central 20-mm segment) and base (20 mm directly above the fascicle sheath).
Sample location
Respiration rate (µmol O2 m –2 s –1)
Ambient (A) (mean ± SEM)
Elevated (E) (mean ± SEM)
Ratio (E/A)
P
Tip
Middle
Base
0.24 ± 0.03
0.35 ± 0.03
0.77 ± 0.12
0.30 ± 0.04
0.30 ± 0.03
0.58 ± 0.09
1.25
0.86
0.75
< 0.01
< 0.01
< 0.01
Mean
0.43 ± 0.02
0.38 ± 0.02
0.88
TREE PHYSIOLOGY ONLINE at http://heronpublishing.com
352
GRIFFIN, ANDERSON, TISSUE, TURNBULL AND WHITEHEAD
trast, the majority of the cells examined from the tip of the
needles were significantly smaller, had relatively more living
cytoplasm, smaller vacuoles and a lower density of mitochondria per unit area of living cytoplasm. Compared with the base,
the apex of the needle would be the most illuminated portion,
and most closely coupled to p(CO2)a, both factors that could
maximize photosynthetic carbon gain. The mid-sections of the
needles were intermediate to the tip and the base, being
slightly larger and more mature than the tip, but not dominated
by transfusion tissue as in the basal sections. Mid-section cells
also had intermediate mitochondrial densities per unit area of
living cytoplasm, nearly twice that of the cells from the tip of
the needles but only a third or less of that at the base of the needles.
Along the length of the needles, respiration per unit area increased from the tip to the base, a pattern that was generally
consistent with the trend in mitochondrial numbers per unit
cytoplasm. One possible explanation for the higher rate of respiration in the base of the needle compared with the tip or
mid-section may be related to phloem loading via the transfusion tissue (Bouma et al. 1995). Other possible explanations
for the higher respiration rate at the base of the needle could be
related to higher nitrogen concentrations leading to higher
maintenance respiration (Ryan 1995), higher cell number per
unit leaf area, or higher concentrations of respiratory substrates (Griffin et al. 2001b). More work is needed to identify
the cause of the higher respiration rates at the base of the needles. Variation in respiration as a function of position within
the needle has practical implications for the measurement of
respiration in coniferous species such as P. radiata, as variation in the location of the sampled tissue would lead to substantial differences in the estimated rate of respiration.
Regardless of the location of the cells (tip, mid or basal sections), needles collected from trees grown in elevated p(CO2)a
had more than twice the number of mitochondria per unit area
of cytoplasm as needles grown in ambient p(CO2)a. This result
is consistent with that of other studies on plants grown in ambient and elevated p(CO2)a (Robertson et al. 1995, Lewis et al.
2000, Griffin et al. 2001a, Tissue et al. 2002). Plant mitochondria are involved in several metabolic processes in addition to
respiration such as the glyoxylate cycle, photorespiration,
nitrogen assimilation, alternative oxidase activity and the
maintenance of the stromal redox state. Together with the
chloroplasts and peroxisomes, mitochondria participate in the
glycolate pathway initiated by the oxygenation of RuBP during photorespiration. We found that the number of peroxisomes per unit area was unaffected by growth in elevated
p(CO2)a, and thus the ratio of peroxisomes to mitochondria decreased. Clearly, the influence of both respiratory and non-respiratory metabolic processes on the response of leaf fine
structure to p(CO2)a treatments requires further study.
Although it is convenient to consider respiration as the active physiological process in the absence of photosynthesis
(i.e., in the dark), it is well known that respiration is required
for photosynthetic carbon fixation (Kromer 1995, Mackenzie
and McIntosh 1999). Furthermore, 25 to 50% of the redox
equivalents produced in plant mitochondria are oxidized else-
where in the cell (Hanning and Heldt 1993). Therefore, Griffin
et al. (2001a) suggested that increased mitochondrial respiration during the day could meet the increased daytime energy
demands of faster-growing, more metabolically active cells,
and therefore partially account for the increased number of mitochondria. Although we did not measure daytime respiration
rates in our needle sections, previous estimates of daytime respiration based on the analysis of photosynthetic response
curves to intercellular CO2 partial pressure at this field site
consistently indicated that daytime respiration rates are higher
for needles grown in elevated p(CO2)a than in ambient p(CO2)a
(Turnbull et al. 1998, Griffin et al. 2000, Tissue et al. 2001), although differences between the CO2 treatments are not always
statistically significant.
In conclusion, mitochondrial number per unit area of living
cytoplasm was significantly higher and nighttime respiration
rates were significantly lower in Pinus radiata needles from
trees grown in elevated p(CO2)a than in needles from trees
grown in ambient p(CO2)a. In addition, both mitochondrial
numbers per unit area of cytoplasm and respiratory activity
varied along the length of the needle, with the highest number
of mitochondria per unit area of cytoplasm and the highest rate
of respiration per unit leaf area at the base of the needle. The
data support our hypothesis that the highest number of mitochondria per unit area of cytoplasm occurs at the base of the
needle (i.e., most mature cells), but the data do not support our
hypothesis that the lowest rate of respiration occurs at the base
of the needle (i.e., lowest cellular growth rate). This may be
because we did not account for the abundance of transfusion
tissue in the base of the needles, and the physiological costs of
phloem loading. We suggest that, although the functional anatomy of conifer needles has rarely been studied, it provides an
excellent system for examining the effects of variations in atmospheric p(CO2)a on foliage structure and physiology during
ontogeny. We have demonstrated that important physiological
and structural differences exist within leaves, as well as between leaves, trees and forests.
Acknowledgments
We thank G.D. Rogers, T.M. McSeveny, J.E. Hunt and J.N. Byers for
their expert help in constructing and maintaining the open-top chamber field site. We also thank Dr. Hans Lambers for improving this
manuscript. This research was supported by the National Science
Foundation, Division of International Programs grant INT-9515449
to DTT and KLG, the National Science Foundation grant IBN
9603940 to KLG and the Packard Foundation Grant DLP 998306 to
KLG and ORA. Majority funding for this field site was provided by
the New Zealand Foundation for Research, Science and Technology.
The research is part of a larger Project 1105 of the Core Research Program for the GTCE (Global Change and Terrestrial Ecosystems) component of IGBP (International Geosphere–Biosphere Program). We
acknowledge the generous loan of the open-top chambers from
USDA Forest Service, North Carolina, USA and assistance provided
by Christchurch City Council, New Zealand Lotteries Grants Board
and Ansett New Zealand and Forest Research. This is LamontDoherty Earth Observatory contribution number 6537.
TREE PHYSIOLOGY VOLUME 24, 2004
FUNCTIONAL ANATOMY AND PHYSIOLOGY OF NEEDLE ONTOGENY
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