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Transcript of Hassan B.Sc., Garyounis - Acadia Uereekie/research/PDF/Lamen, MSC1998.pdf · 2009. 2. 2. · Effect...
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Effect of eleva ted carbon dioxide on seedling morphology and anatomy in seven herbaceous species
Sabah Hassan Lamen B.Sc., Garyounis University, Benghazi-Libya, 1989
Submitted in partial fulfillrnent of the requirement for the Degree of Master of Science (Biology)
Acadia University Spring Convocation 19%
O by Sabah Hassan Lamen, 1998
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Table of Contents
....................................................................... List of tables .v
...................................................................... List of figures vi
. . - ............................................................................. Abstract v m
................................................................ Acknowledgments .x
........................................................................ Introduction - 1
........................................................... Materials and methods 9
.............................................................................. Results 13
......................................................................... Discussion 28
......................................................................... Re ferences 3 4
........................................................................ Appendices 40
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List of Tables
Table 1. The level of significance for the effect of carbon dioxide on
matornical and morphological characteristics of seven species
.... ... ................................................................ 25
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List of Figures
Figure 1. The effect of CO, on stem diameter and parenchyma ce11 width (A) and on
stem height and ce11 length (B) in Sinupis aha. In the case of cell width
(A). enor bars (*l SE) are not shown because they were smaller than the
size of the symbol, whereas error bars for stem height are not s h o w
because the data were not normally distributed ........................... 15
Figure 2. The effect of CO1 on stem height of six genera ........................... 16
Figure 3. The effect of CO, on stomatal and epidermal ce11 density of Sinupis albrr.
In the case of epidemal cell density on upper surface. error bars (d SE)
are not shown because the data were not normally distributed. whereas for
the other cases error bars were srnaller than the size of the
symbol.. ........................................................................... -17
Figure 4. The effect of CO2 on stomatal and epidermal ce11 density of six genera.
Symbols as in figure 3. In the case of stomatal density on Dianthus lower
surface and epidermal ce11 density on Beta upper surface, error bars
(h 1 SE) are not shown because the data were not normally distributed.
whereas for the other cases error bars were smaller than the size of the
........................................................................... symbol.. -18
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vii
Figure 5. Scanning Electron Micrograph (SEM) of the lower cotyledon surface of
Sinapis alba grown at 350 ppm CO,. Scale bar = 10 pm.. ............... .A9
Figure 6. Scanning Electron Micrograph (SEM) of the lower cotyledon surface of
.................. Sinapis albu grown at 700 ppm CO,. Scale bar = 10 pm.. 20
Figure 7. Scanning Electron Micrograph (SEM) of the lower cotyledon surface of
Sinapis alba grown at 1400 ppm CO,. Scale bar = 10 m.. .................. -2 1
Figure 8. The effect of CO? on stomatal size in Sinapis alba on both the upper (A)
and the lower cotyledon surfaces (B). In the case of length of guard cells
on lower surfaces, error bars (*ISE) are not shown because the data were
not normally distributed. whereas for the other cases error bars were
............................................. smaller than the size of the symbol 22
Figure 9. The effect of CO, on size of stomata in the upper surface of six genera.
Symbols as in figure 8. In the case of length and width of guard cells in
Cuctmis and Beta, and in length and width of subsidiary cells in
Raphanzrs, error bars ( i l SE) are not shown because the data were not
normally distributed, whereas for the other cases error bars were smaller
................................................ than the size of the symbol.. ..23
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Figure 10. The effect of CO2 on size of stomata in the lower surface of six genera.
Symbols as in figure 8. In the case of length and width of both guard and
subsidiary cells in Medicago, width of guard cells in Cucumis, Beta and
Raphanus. and length and width of guard cells in Celosia. error bars
(* 1 SE) are not shown because the data were not normally distributed.
whereas for the other cases error bars were smaller than the size of the
symbol.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .-.-. . . . ..24
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ABSTRACT
The eRects of carbon dioxide on stomatal density, stomatal index. stomatal size.
and epidermal ce11 density were exarnined in seven genera (Sinapis albu L.. Medic~rgo
suriva L ., Ceiosiu cristata L . 'plzirnosus ' , Dianlhus caryophyllzis L . , Cucumis sur ivzcz L ..
Beta nvulgaris L. var. cicla L.. and Raphanus sarivus L.) using scanning electron
microscopy. In the case of Sinapis only, stem diameter, and the length and width of
cortical parenchyma cells were exarnined using light microscopy. Stem length was
measured in al1 the studied species. Plants were grown in growth chambers maintained at
IOC and at one of three Ievels of carbon dioxide (350.700. and 1400k50 pprn). Seedlings
were provided with enough light to induce the unfolding of the cotyledons but not enough
to result in any significant photosynthesis (PAR45 pmol m" s-' for 10 minutes per day).
Stem elongation in Sinapis and Medicago. lengths of both guard and subsidiary-ce11 in
Sinupis. and lengths of parenchyma cells were ail significantly increased by elevated
carbon dioxide. In contrast. stomatal density and epidermal ce11 density in Sinapis and
Medicugo were decreased for both cotyledon surfaces. Stomatal density on the lower
surfaces of Celosiu and Beta was also significantly decreased by elevated carbon dioxide.
Level of carbon dioxide had no effect on stem diameter, diameter of parenchyma cells or
on stomatal index in any of the species. Level of carbon dioxide did not significantly
affect any of the measured parameters in either Diunthzrs or Raphanus. The results
suggest that elevated carbon dioxide increases longitudinal ce11 expansion and that this
has various consequences such as a decrease in stomatal density and increases in stem
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height. Furthemore. the effect of carbon dioxide on ce11 expansion is species specific and
independent of its eEects on photosynthesis.
Key words: elevated carbon dioxide, phytochrome. stomatal density. stomatal index.
epidermal ce11 density. stomatal size and scanning electron microscopy.
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Acknowledgments
1 would like to thank my supervisor Dr. E.G.Reekie for his guidance.
encouragement and help in al1 aspects and al1 stages of the thesis. 1 would like to thank
my Co-supervisor Dr. H.Taylor for her support and patience with the scanning electron
microscope (SEM). and to Dr. D. Knstie for his continued support and being there when 1
nreded him. 1 would also like to thank Dr. G. Gibson and H. Smith for their help in the
preparation of specimens for electron microscopy. Thanks also to K. Zebian. H. Stone.
M. Ntiarnua and to R. Newell for technical assistance. I would like to thank the
secretaries in Biology. Jeanette Simpson and Wanda Langley for their assistance and
patience. 1 am forever in debt to Cnends particularly J.Y.C Reekie. H. Hewlin. H.N.
Lantz. 1. Wong and N. Musa for their support and understanding. A great big thank you to
my dear husband Awad E. Elsharey for his patience. suppon and love. A special thanks to
my country Libya who sponsored my studies in Canada and especially to my parents to
whom I dedicate this thesis.
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INTRODUCTION
There is no doubt that atmospheric levels of carbon dioxide have been nsing and
that this rise is chiefly due to fuel combustion and deforestation. At present. there are 350
ppm carbon dioxide in the atmosphere. It has been predicted that this concentration will
double to about 700 ppm by the last half of the 2 1 '' century (Bazzaz. 1990: Bazzaz and
Fajer. 1 992: Wittwer. 1 992). Therefore. researchers have shown a great deal of interes t in
the effects of CO2 on plant growth (Stuiver. 1978; Woodwell. 1978). information on plant
responses to CO2 enrichment may help to forecast consequences for plant productivity.
cornmunity composition, and ecosystem structure (Jarvis. 1987).
It is generally accepted that the pnmary effects of increasing CO, concentration
are on photosynthesis and stomatal conductance: photosynthesis is increased and stomatal
conductance is decreased. Increases in photosynthesis are due to the beneficial effects of
CO, concentration on carboxylation efficiency and the consequent decrease in
photorespiration. Decreases in stomatal conductance are due to the effect of interna1 CO,
upon stomatal closure (Bazzaz. 1990) though the rnechanism by which CO2 controls
stomatal activities is not known (Pearcy and Bjorkman. 1983). This decline in stomatzl
conductance leads to a reduction in water loss and consequently results in the
enhancement of plant water use efficiency (defined as the nurnber of units of CO, tïsed in
a given time per unit of water lost in the same time period).
Although the major effects of elevated CO? on plants are on photosynthesis and
stomatal conductance, it modifies a broad range of plant processes including many
aspects of development. such as: leaf morphology and anatomy, biomass allocation. time
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of flowering, rate of senescence. branching patterns and stem elongation (Strain and Cure.
1985: Eamus and Jarvis. 1989; Bazzaz, I W O ; Zebian and Reekie. 1998). The effects of
CO, upon development can be complicated: for example. depending upon species and
environmental conditions, elevated CO, rnay delay? hasten. or have no effect on tirne of
flowering (Reekie and Bauaz. 199 1). In general. developmental effects such as these are
considered secondary consequences of the effect of CO, on photosynthesis and stomatal
conductance and therefore on growth. However. the effects of COZ on phenology are not
necessarily correlated with its effects on growth (Reekie and Bauaz. 199 1). Furthemore.
it has been shown that CO, can affect stem elongation in complete darkness (Le. in the
absence of any of its effects on photosynthesis or growth) (Zebian and Reekie. 1998).
One aspect of the effect of CO, on development is a change in stomatal density
and rnorphology. Changes in stomatal density (defined as the number of stomata per unit
of interveinal leaf area (Salisbury. 1927)) and the length. width. or area of stomatal
apertures may contribute to the reported increases in water use eficiency with elevated
COz. There are three lines of evidence indicating thai stomatal density changes with CO2
concentration: ( 1 ) histoncal studies (Le. fossil leaves and herbarium marerial). (2)
altitudinal studies and (3) controlled environrnent chambers. Each approac h has iis onn
advantages and disadvantages.
Historical studies:
Fossil and old herbarium specimens attracted the attention of scientists because of
the documented change in CO? concentration over time. There fore. several scientists have
examined fossil leaves and herbarium materiai to determine if stomatal density has also
changed over time (Woodward. 1987; McElwain et al.. 1995: Pefiuelas and Matamala.
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1990; Beerling et al.. 1992). The first three studies have found that stomatal density
decreases with time and therefore, with increasing COz concentration. However. the
fourth study (Beerling et al. 1992) found a different trend in that stomatal density also
increases as CO, concentration increases. These changes in stomatal density could be the
consequence of either acclimatory or adaptive responses. In this regard. the timr scale
involved may be significant. By comparing the Woodward ( 1987) and Beerling er UL .
(1 992) studies. it is clear that natural selection over the 1 1 500 year period concemed in
the Beerling et al. study may have favoured a different response to what is. in effect. an
acclirnatory response noticed in trees within the period of rapid COz rise of the past 200
years in the Woodward study. Given this time span in the Woodward study. it is unlikrly
the change in stomatal density represents any genetic change.
The study of stomatal characters in fossil leaves and herbaium material does not
consider either variation in variables other than COz over time. or artifacts introduced by
fossilization and specimen collection. Therefore. it has been argued that conclusions
drawn from counts of stomata from fossil materiai are of limited value and must be
treated with extreme caution (Poole et al.. 1996).
Altitudinal studies:
It has been documented that stomatal density generally increases with altitude. As
altitude increases, the COz mole fraction remains constant but the partial pressure of CO2
decreases (Gale. 1972). This suggests that the nse in stomatal density with altitude rnay
be related to CO? partial pressure. However, stomatal density is influenced by a nurnber
of factors in addition to COz such as: light intensity, water availability, nutrient
availability. and temperature (Salisbury and Ross. 1992). Given that al1 these factors also
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Vary with altitude, the reported response of stomatal density to altitude may therefore be
the product of a combination of environmental factors (e-g. CO2 levels. temperature. light.
water availability). For example. the influence of temperature on changes in the stomatal
density of plants with altitude becomes increasingly difficult to differentiate because of
the effects of changes in temperature and atrnosphenc CO2 concentration. which widely
MI with altitude, and irradiance which increases with altitude (Korner et al.. 1989). This
is a major disadvantage of this sort of study. It should be noted. however. that Woodward
( 1986) has shown experimentally that observations of increasing stomatal density with
altitude in the field can be simulated in the laboratory by changing CO, mole fraction. at a
constant atmospheric pressure. In another laboratory experiment. Woodward and B a u a z
( 1988) simulated the effect of altitude on CO2 partial pressure. by examining the
sensitivity of stomatal density to changes in CO2 partial pressure. at a stable mole
haction. These authors found that al1 of the species under study had the ability to respond
to a reduction in the partial pressure of CO, by an increase in stornatal density.
Controlled environment chambers studies:
Many researchers have attempted to investigate changes in stomatal density with
CO, concentration by growing plants under both ambient and elevated CO, in controlled
environment chambers (O'Leary and Knecht. 198 1 : Woodward. 1987: Woodward and
B a v a t 1988: Radoglou and Jarvis. 1990; Radoglou and Jarvis. 1992; Malone et al..
1993; Ceulemans et al.. 1995; Famsworth et al.. 1996). One disadvantage of growing
plants under controlled environments is that plants are exposed to immediate increases in
CO, concentration whereas. in nature. plants are subjected to graduaf fluctuations in the
atmospheric COZ concentration. Thus. this might affect the responses of stomatal density
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to COz. Criticisms have been made of experiments exposing plants to elevated COZ
regimes in smail scale giasshouses and field enclosures because of the effects of field
enclosures upon wind speed and hurnidity and their subsequent effects on the balance
between energy supply and water loss from leaves (Morison. 1987). In addition. under
controlled environment conditions. plants are grown under controlled CO2 concentration
while under a lower irradiance or different spectral distribution than that which occurs
naturally. Both CO2 and light cause acclirnatory responses in plants. nius. for predictive
aims. it would be preferable to control the CO, concentration over most or al1 of the life
cycle. and use natural solar irradiance (Rowland-Barnford et ai.. 1990).
Conclusions of previous studies:
Evidence provided from these studies indicate that there is a great deal of
variation in the response of stomatal density to CO. concentration. Some of this variation
ma- be attributed to differences in growth conditions arnong studies. For instance. most
of the growth chamber studies were short-tem. enduring for days. weeks or months and
rrpresent only a short-term acclimatory response rather than any long-term phpsiological
adaptation (Jarvis. 1989). On the other hand. studies that deai wi th changes of stomatal
density with altitude and over time, rnay represent either acclimatory or adaptive response
or both.
From al1 of the previous studies. it is clear that the response of stomatal density to
COz varies extensively arnong species and cultivars. Also. stomatal density on both
abaxial and adaxial surfaces of the same leaf may respond differently to CO2
concentration (O'Leary and Knecht. 198 1 ; Rowland-Barnford ef ai.. 1990; Ceulemans et
(11.. 1995). For example. in the Rowland-Bamford ei al.. ( 1990) study. ihere was a
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different effect of COL on the two leaf surfaces. the cause of which is unknown. The
greatest effect of CO2 on stornatal density occurred on the abaxial surface of the fag
leaves. At the higher CO, values this resulted in the abaxial surface having a greater
stomatal density than the adaxial surface.
Several studies have attempted to determine whether the effect of COz on stomatal
density is real or an artifact due to increases in ce11 size. For instance. if the size of the
epidemal cells increases, the total nurnber of stomata per unit area will decrease.
Consequently. many studies have determined stomatal indes (defined as the ratio of
stomatal cells to epidermal cells expressed as a percentage) dong with stomatal density.
Some studies (Woodward, 1987; Beerling et al.. 1992: Ceulemans eï ul.. 1995: McElwain
et al.. 1995: Poole et al.. 1996). conclude that the effect of CO, on stomatal density is real
(Le. on stomatal development) in that stomatal index changed in concert with stomatal
density. Whereas. other studies. (e-g. Farnsworth el al.. 1996) however. ascnbe the
decreases in stomatal density to epidermal ce11 enlargement in elevated CO' rather than to
the production of additional stornata under ambient CO:. Still other studies (Radoglou
and Jarvis. 1990: Radoglou and Jarvis. 19%: 1992: Malone et al.. 1993: Lauber and
Komer. 1997). conclude that the lack of differences in both stomatal density and stornatal
index with CO, supports the hypothesis that there are no direct effects of elevated CO, on
the initiation of stomata during ontogenesis or on epidermal cell expansion at a later
stage.
Not all of the studies dealing with the response of stomatal charactenstics to CO,
use the same range of CO2 treatrnents. For exarnple, some studies use subarnbient CO,
concentrations (Woodward. 1 987; Woodward and Bazzaz, 1 988; Rowtand-Bamford et
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al.. 1990: Malone et al.. 1993) and some use superambient (O' Leary and Knecht. 198 1 :
Rowland-Barnford et al.. 1990; Radoglou and Jarvis, 1 990; Ryle and Stanley. 1 992) CO,
concentration. Generally. there is a significant effect of COz on stomatal density at
subambient CO? concentrations but some studies show that as COz concentration
increases above arnbient there is little or no eRect of CO, on stomatal density (Woodward
and Bazzaz, 1988: Rowland-Bamford et al.. 1990). For example. Woodward and Bazzaz
( 1988) have shown that stomatal density and stomatal index decreased as the
concentration of CO, increased fiom the pre-industrial concentration to the present
concentration. but decreased only slightly when the concentration of CO, was funher
increased to double the present concentration.
Objectives of present studv:
As mentioned earlier. the effects of CO, on developmental patterns are considered
secondary consequences of the effects of CO2 on photosynthesis and therefore on growth.
Several studies however. suggest this interpretation may be too simplistic and that CO,
concentration ma? have direct effects on development (Reekie and Bazzaz. 199 1 : Zebian
and Reekie. 1998). Because the change in stomatal density is another aspect of
development. it could be argued that the effects of CO, on stomatal density and
rnorphology are also independent of its effects on photosynthesis and growth. The present
study was undertaken to determine if carbon dioxide can affect stomatal density in the
absence of light and therefore in the absence of any effects on photosynthesis.
Seven genera of plants (Sinapis alba L. (Brassicaceae). iMedicago surivu L.
(Fabaceae). Celosia cristata L . -plrirnoszrs' ( Amaranthaceae), Dianthus caryophyiizds L .
( Cary ophy llaceae), Cucumis sativus L . (Cuc urbitaceae), Beta vulgaris var. c icla L .
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(Chenopodiaceae). and Raphanur sativus L.(Brassicaceae) were gro wn at one of three
levels of carbon dioxide. Seedlings were provided with enough light to induce the
unfolding of the cotyledons but not enough to result in any significant photosynthesis.
We exarnined the effect of these treatments on stem height, stomatai density. stomatal
index. stomatal size. and epidermal ce11 density in al1 the species. Stem diameter and
Iength and width of parenchyma cells were measured in Sinupis alone. We hypothesized
that if CO, c m affect stomatal density in the absence of light and therefore. in the absence
of any effects on growth. this will indicate that CO, affects stomatal characteristics by
sorne mechanism(s) other than through its effects on growth. We also hypothesized that if
stomatal index does not change with level of carbon dioxide. this will indicate that both
the reported decreases in stomatal density and increases in stem height at elevated CO,
can be attributed to simple increases in ceIl size and therefore decreases in number of
cells per unit area.
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Materials and Methods
Carbon Dioxide Fumigation System
The seedlings were exposed to the different CO, concentrations (350. 700. and
1400 ppm) using a flow through fumigation system. The himigation chambers (0.75 L )
consisted of an 11 cm length of a 10 cm diarneter PVC pipe with an opaque cap on one
end and a removable transparent plexiglass cover on the other. Each charnber had a 0.5
cm diarneter inlet tube on one side and a 0.7 cm diarneter outlet tube on the other. The
outlet tube was comected to a 30 cm length of Tygon tubing to minirnize back diffüsion
into the charnber. The inlet tube of each chamber was coupled to a humidified air supply
systern which provided a flow of I L per minute per charnber. The air was obtained from a
pressure regulated compressed air source that sampled air from outside the buildin, u at a
height of about 2m above ground level. The 350 ppm CO, treatment was unmodified
arnbient air. The 700 and 1400 pprn CO, treatments were obtained by bleeding pure CO:
into the atmospheric air source via needle valves. The level of CO, in the various
airstreams was determined using a Li-Cor mode1 6250 infrared gas analyser. hl1
fumigation charnbers were placed in the sarne controlled environment charnber (Conviron
mode1 E 15) which maintained a constant temperature of ZOC.
Plant culture
In the expenment involving Sinapis, 15 fumigation chambers were used. five at
each of three carbon dioxide levels (350,700. and 1400 ppm). However. with al1 the
other species, 18 fûmigation chambers were used. six at each of the three CO, levels. Al1
seven species were grown fiom seed in 50 ml vials containing 0.6% agar. A wick was
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placed in the agar with both ends protmding through holes thot were dnlled in the sides
of the viais. The vials were then placed in the fumigation charnbers that contained about
60 ml distilled water. The wicks absorbed water thus keeping the agar surface moist. Ten
seeds were evenly spread upon the surface of the agar in each vial. There was a total of 30
Sinupis seedlings per chamber (Le. 3 vials per chamber). In the experiments involving the
other species. each fumigation charnber contained ten seedl ings of each species. Although
the expenmental conditions were the same. due to differences in germination rates. the
species were harvested at different times: Sinupis-6 days. Medicago- 1 1 days. Diunrhzis- 13
days. CeIosiu-15 days. Raphanus-7 days. Beru and Cucumis-9 days.
Experirnental Measurements
Plants were hanested. stem height recorded and samples stored in formalin-
aceto- alcohol ( F M ) solution. To prepare samples for Scanning Electron Microscopy
(SEM). one cotyledon per charnber per treatrnent was fixed in 2.5% Glutaraldehyde in
PO, buffer for 2-4 hours. Afier 3 successive washes in 0.1 M PO, buffer. the samples
were then postfixed in 1% OsO, in O. 1 M PO, buffer for 1 hour. The sarnples were
\vashed twice with 0.1 M PO, buffer and dehydrated through a graded acetone series ( 15.
30.50. 70. 80.90.95%) for 10 minutes per concentration. Three changes of 100%
acetone completed the process. Critical point drying from the acetone followed. The
required solutions were prepared as descnbed in Appendix 1. Appendix 2 provides a
detailed protocol for the fixation procedure. The cotyledons were mounted on stubs and
spuner coated with 15-20 nm gold-palladium. The cotyledon surfaces were viewed at 15
KV on a JSM 25s. Stomatal density measurements were recorded by counts made on
screen. A field of view on the cotyledon surface located away from the midrib region was
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chosen randomly by moving the sample on both the X and Y axes. The total number of
stomata (per field of view at 450X) was counted. This procedure was repeated for both
the lower and upper surfaces for each cotyledon.
Lengths and widths of both guard and subsidiary cells were measured from
Scanning electron micrographs (at 1000X). Al1 stomata visible on the photograph were
measured. These photographs were also used to measure epidermal ce11 density by
counting the total number of the epidermal cells visible on the photograph. Data fiom one
cotyledon per chamber were recorded. Stomatal index was calculated as: number of
stomata per unit area / (nurnber of stomata + nurnber of epidermal cells per unit
area)x 100.
In the case of Sinapis only. light microscopy was used to measure both the length
and width of parenchyma cells and the diameter of the stem. FAA fixed stems were cut
0.5 cm below the cotyledons into sections not longer than 0.5 cm. The sections were
dehydrated using a Tertiary Butyl Alcohol (TBA) series followed by infiltration in
paraffin wax The sarnples were then embedded in hot paraffin. The resulting paraffin
blocks were trimrned and mounted ont0 wooden blocks. Blocks were sectioned (30prn)
both transversely and longitudinally and sections mounted on g l a s slides (Appendix 3).
Observations were made at 16X and the dimeter of the stems recorded from the
transverse sections. For measurements of length and width of cortical parenchyrna cells.
the longitudinal sections were stained with fast green (Appendix 4). Observations were
made of ce11 length and width at 40X. A row of cells located midway between the
epidermis and the edge of the vascular cylinder was chosen for measurement. Frorn this
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row the dimensions of 5 adjacent cells per stem were recorded with 5 stems per treatment
(Le. one stem /chamber).
Statistical analysis
These data were analyzed using one-way ANOVA for nomally distributed
variables wiih COZ as the independent variable and Kmskal-Wallis non-parametric tests
for non-normally distributed variables. Al1 analyses were done using Sigma Stat (Version
1 .O 1 ) for a persona1 cornputer.
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Results
Level of carbon dioxide had no effect on stem diameter nor upon width of stem
parenchyma cells in Sinapis (fig 1 A. table 1 ). However. it had a pronounced effect on
stem height and length of stem parenchyma cells (fig lB, table 1). Level of carbon
dioxide also had a pronounced effect on stem height in Medicago. Level of carbon
dioxide did not significantly affect stem height in any of the other species (fig 2. table 1 ).
Stomatal frequencies were lower on the upper cotyledon surfaces in Sinupis.
.bledicugo. and Betu. In al1 the other species. there was roughly an equal number of
stomata on both the upper and lower surfaces. Stomatal and epidermal ce11 density in
Sinapis (tigs 3 . 5. 6 and 7. table 1 ) and Medicago (fig 4. table 1 ) were significantly
decreased by elevated carbon dioxide on both the upper and lower cotyledon surfaces.
However. in Celosia and Beta. stomatal density was only decreased on the lower
cotyledon surfaces (fig 4. table 1). In the case of Dianrhus and Ruphumrs. level of carbon
dioxide had no effect on either stomatal or epidermal cell density (fig 4, table 1 ).
Epidrrmal ce11 density on the lower cotyledon surface of Cdosiu was also significantly
decreased (fig 4. table 1). Stomatal index (defined as the ratio of stomatal cells 10
epidermal cells expressed as a percentage) was not affected by level of carbon dioxide in
any of the species.
In the case of Sinapis only, level of carbon dioxide caused a significant increase in
the length of both guard and subsidiary cells (figs 5,6.7 and 8. table 1). However. level
of carbon dioxide had no effect upon width of both guard and subsidiary cells. Level of
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carbon dioxide had no effect on stornatal size on either the upper (fig 9, table 1 ) or Iower
( fig 10. table 1 ) cotyledon surface in any of the other species.
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Stem diameter fi Ce11 width
-- - - - --
0 Stem height Cell length
350 700 1400 CO2 level (ppm)
Fig. 1. The effect of CO7 - on stem diarneter and parenchyma ce11 width (A) and on stem height and ce11 length (B) in Sinapis albo. In the case of ce11 width (A), error bars (*l SE) are not shown because they were srnaller than the size of the symbol. whereas error bars for stem height (B) are not shown because the data were not normally distributed.
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Medicago
CO2 level (ppm)
Fig. 2. The effect of CO2 on stem height of six genera.
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Stomatal density on upper surface
Stomatal density on lower surface
Epidermal ce11 density on upper surface
Epidermal cell density on Iower surface
CO2 level (ppm)
Fig. 3. The effect of CO? - on stomatal and epidermai ce11 density of Sinapis albu. In the case of epidermal ce11 density on upper surface. error bars (* I SE) are not s h o w because the data were not normally distributed. whereas for the other cases error bars were smdler than the size of the symbol.
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Medicago x
Raphanus
O 350 700 1400
level (ppm) Fig. 4. The effect o f CO2 on stomatal and epidermal ce11 density of six genera. Symbols as in figure 3. In the case of stomatal density on Dianthus lower surface and epidermal ce11 density on Beta upper surface, error bars (*ISE) are not shown because the data were not normally distributed, whereas for the other cases error bars were smaller than the size of the symbol.
-
Fig. 5. Scanning Electron Microçraph (SEM) of the lower cotyledon surface of Sinupis d b a grown at 350 ppm CO?. - Scale bar = I O pm.
-
Fig. 6 . Scanning Electron Micrograph (SEM) of the lower cotyledon surface of Sinapis alba grown at 700 ppm CO7. - Scale bar = 10 pm.
-
Fig. 7. Scanning Electron Micrograph (SEM) of the lower cotyledon surface of Sinapis ulba grown at 1400 ppm CO-. Scale bar = IO Pm.
-
(A) - ..
Length of guard ceiis a Width of guard cells
Length of subsidiary cells O Width of subsidiary celIs
Length of guard ceils - Width of guard cells 0 Length of subsidiary cells - 0 Width of subsidiary cells
I - m
II m m
CO2 level (ppm) Fig. 8. The effect of CO7 - on stomatal size in Sinapis alba on both the upper (A) and the lower cotyledon surfaces (B). In the case of length of guard cells on lower surfaces, error bars ( i l SE) are not shown because the data were not normally distributed, whereas for the other cases error bars were srnaller than the size of the symbol.
-
evel (ppm) Fig. 9. The effect of CO2 on size of stomata in the upper surface of six genera. Symbols as in figure 8. In the case of length and width of guard cells in Cucumis and Beta, and in length and width of subsidiary cells in Ruphanus, error bars (* 1 SE) are not s h o w because the data were not normally distributed, whereas for the other cases error bars were smaller than the size of the symbol.
-
Raphanus
CO2 level (ppm) Fig. 10. The effect of CO? - on size of stomata in the lower surface of sis genera. Symbols as in figure 8. In the case of length and width of both guard and subsidiary cells in Medicago, width of guard cells in Cucumis, Beta and Raphanus, and length and width of guard cells in Celosirr. error bars (*l SE) are not shown because the data were not normally distributed. whereas for the other cases error bars were srnaller than the size of the symbol.
-
Table 1. The level of significance for the ellèçt of carbon dioxide on unaiornical and tnorpliological characteristics
of seven species. Unless otlierwise iioted, level of significance was based on one-way analysis of variance.
Plant species
Variable Sir tupi?; Meclicqo Dirr I I t h us Celosiu Raphcrnrts C'trczrmis Beta
Stomatal density
Upper surface ~ 0 . 0 1
Lower surface KO.0 1
Epiderrnal ceIl density
Upper surface
-
Discussion
Our results have confirmed earlier studies that found that the effects of CO, upon
development are not necessarily mediated through photosynthesis (Reekie and Bazzaz.
199 1 ; Zebian and Reekie. 1998). For instance. in our study, the fact that carbon dioxide
affected stem elongation and stomatal density at extremely low light levels (Le. in the
absence of any of its effects on photosynthesis) confirms that the effect of CO, on
development is not necessarily related to its effect on photosynthesis.
The results concrrning Sinapis and Medicago. suggest that elevated CO2 increases
ce11 expansion and that this has various consequences such as a decrease in stomatal
density and increase in stem height. The fact that stomatal index does not change wiih
level of CO, indicates that the decrease in stomatal density at elevated CO, can be
entirely attributed to simple increases in ce11 size and therefore decreases in numbers of
cells per unit area. Similarly. the 26% increase in Sinapis height between 350 and 1400
pprn CO2 can be more than accounted for by the 66% increase in length of stem cells. It
would be usefùl to view cells taken fiom other regions of the stem besides those used in
this study (Le. fiom the mid-point of stem). By examining cells from the base to the apex
one should be able to determine both a) the proportion of stem cells that shows this
degree of expansion. and b) whether total ce11 number is reduced which indicates a
reduction in ce11 division. The effect of CO, on these two divergent aspects of plant
development (i.e. stomatal density and stem elongation) appears to be the result of the
effect of CO, on a single process, ce11 expansion.
-
The results also suggest that the effect of elevated CO2 on ce11 expansion is
variable depending upon species. plant tissue and ce11 type. There was no effect of CO,
on stem height in any of the remaining species and no effect on stomatai or epidermal ceIl
density in Dianthus and Raphanus. Also. there was no effect of COz on stomatal size in
any of the species except for Sinnpk. Elevated CO, did decrease stomatal density on the
lower cotyledon surface of Ceiosia and Betu. but not on the upper surface. The decrease
in stomatal density in both species was accompanied by decreases in epidermal ce11
density. but these decreases were only significant at the 0.05 level in the case of Cdosicr.
However. in none of these cases was stomatal index affected by CO,. again suggesting
that the effect of CO, on stomatal density was simply due to effects on ce11 expansion. In
the case of those species and tissues where COz did not affect ce11 expansion. it would be
interesting to examine the effects of subambient CO, levels. Previous studies suggest that
subarnbient CO2 levels have a greater effect on stomatal density than superambient levels
(Woodward and Bazzaz. 1988: Rowland-Bamford et ai.. 1990).
Greater ce11 expansion, which occurs at higher CO, levels, has been attributed to
the increased osmotic potential of leaf cells associated with rheir higher total
carbohydrate content. which causes the cells to absorb more water and thus enlarge
(Madsen. 1973). However. Our observations suggest that the effect of CO2 on ceIl size
was not dependent on its effects on photosynthesis. Plants were not provided with
sufficient light to result in any significant photosynthesis. Therefore, the increase in ce11
size cannot be attributed to the positive effect of elevated CO2 on carbohydrate
accumulation. This raises the question of how CO, affects ce11 expansion if not through
effects on photosynthesis.
-
One possible mechanism is an interaction between COZ and plant growth
regulaton such as ethylene. At high concentrations (50.000-100,000 p1 I"), CO2 interferes
with the role of ethylene by affecting its synthesis. High levels of CO, decrease the
conversion of ACC ( 1 -Arninocyclopropan- l -Carboxylic acid) to ethylene by acting as a
cornpetitive inhibitor (Cheveny et al. 1988). If the levels of CO2 used in our study
decreased ethylene production. this could explain the increased stem elongation at high
CO, levels as ethylene inhibits stem elongation through its inhibitory effect on cell
elongation (Camp and Wickliff. 1 98 1 : Eisinger. 1 983). This could rxplain the effect of
CO2 on the size of epidermal celis. Although the levels of carbon dioxide used in our
study were much lower than those generally reported to inhibit ethylene production. at
least one study has s h o w that a doubling of ambient CO, levels can affect rthylene
production in some species (Lu and Kirkharn. 1992).
Another possible mechanism by which CO1 may affect ce11 expansion is an
interaction between CO, and phytochrome (the pigment that controls photornorphogenic
responses). Phytochrome. by detecting the presence and spectral quality of light (i.e.
red/far-red ratio). is known to affect a wide range of developmental processes including
flowering. branching patterns. leaf development. seed germination and stem elongation.
Phytochrome exists in two forms: a red-absorbing form called Pr and a far-red absorbing
form called Pfi. Absorption of red light by Pr converts the pigment to the far-red
absorbing form while subsequent absorption of far-red light by Pfi drives the pigment
back to the red-absorbing form. The Pfr form also decornposes slowly to the Pr form in
darkness in a reaction called dark reversion. This reversion has a regulatory function.
-
particularly in relation to seed germination and flowering (Hopkins. 1995). For exarnple.
it is used by seeds to detect whether they are on the surface of the soil or beneath the soil
surface. Also. by sensing the length of the day. plants are able to recognize the growing
season and control their flowenng. The presence of a recüfar-red reversibility provides
information that helps plants to acclimate to their light environment. The redfar-red ratio
varies notably in different environments. At the bottom of the canopy. there is a lower
redfar-red ratio due to the absorption of red light by chlorophyll than closer to the top of
the canopy (Le. high redffar-red ratio). Thus. phytochrome c m f i c t i on as an indicator of
the degree of shading. As shading increases. RER decreases and the ratio of Pfr to the
total phytochrome (Le. PfrlP,,,) decreases. This is used to help plants to control various
changes in morphology (e-g. leaf anatomy. branching patterns and stem elongation).
which aid plants in acclimating to different environments.
An interaction between phytochrome and CO, has been suggested by several
previous studies to explain the contrasting effects of CO2 on flowering and development
under long versus short day conditions (Pumhit and Tregunna 1974: Hicklenton and
Jolliffe. 1980: Reekie et ui.. 1 994: Reekie cl al.. 1997). Further. Zebian and Reekie
( 1998) suggest the contrasting effects of COz on stem elongation in darkness versus light
rnay also be related to an interaction with phytochrome. The effects of phytochrome on
stem elongation have been shown to be pnmarily the result of changes in ce11 expansion.
though in some cases ce11 division is also af5ected (Gaba and Black, 1983). Therefore an
interaction between CO, and phytochrome might explain the effects of COz on ce11
expansion in the present study. None of these studies however, have been able to offer a
mechanism which would explain such an interaction between CO, and phytochrome.
-
A third possible mechanism by which CO2 may affect ce11 expansion is via
acidification (Le. the so called acid growth theory). This theory States that auxin (indole-
3-acetic acid. IAA) initiates an active acidification mechanism. possibly a membrane
bound proton pump. so that pH of the wall solution decreases. Consequentiy. this lower
pH activates wall-loosening enzymes. the wall is loosened. and ce11 enlargement takes
place (Rayle and Cleland. 1970). When COZ dissolves in water it fonns carbonic acid.
which dissociates into bicarbonate and hydrogen ions. which in tum lower the pH. This
acidic pH would promote cell enlargement. It has in fact been suggested that the effect of
IAA on ce11 expansion is mediated through this CO2 effect. Sloane and Sadava. ( 1973)
suggest that IAA may increase cell respiration which in tum releases CO, and lowers the
pH of the ceil wall. However. it is unclear whether the levels of CO2 used in the present
study would be high enough to lower the pH of the ceil wall sufficiently. At least one
previous study has shown that a CO2 concentration of 5% is required to induce significant
ce11 elongation (Mer and Richards. 1950). However. different studies have shown
diffèrent values for the pH required to initiate ce11 wall loosening and extension growth
(Vanderhoef and Dute. 198 1). These values range fiom 6 to 4. Such variation might
explain the contrasting effects of CO2 on different species. plant tissues and ce11 types
observed in the present study.
Ecological implications of the effect of CO, - on stomatal density:
Research has shown that the effects of carbon dioxide on growth are highly
variable. Whether or not enhancement occurs and if it does, the degree of enhancement
varies extensively depending on species and environmentai factors such as nutrients.
temperature. water and irradiance (Bauaz. 1990: Reekie and Bazzaz, 199 1 ; Bazzaz and
-
Fajer, 1992; Poorter, 1993). Also, these effects are ofien transitory (Le. plants may
acclimate such that the response is decreased over tirne). This phenornenon is important
since it will ultimately determine the impact of changes in CO2 on plant productivity.
Previous studies have largely attributed this decline in CO, response to feedback
inhibition on photosynthesis. for instance, excess carbohydrate accumulation and the
consequent darnage to the chioroplast structure (Bazzaz? 1990). However. direct effects of
CO2 on developmental processes such as described here, also have the potential to explain
changes in response over time. For example. decreases in stomatal density at elevated
COZ may decrease stomatal conductance providing the associated increases in stomatal
size do not fully compensate for the decrease in density. This in turn, rnay decrease
photosynthesis (see aiso Reekie et al.. 1997). Therefore. it is important to understand why
CO2 affects developmental patterns if we are going to accurately predict the effect of
changes in the concentration of atrnospheric carbon dioxide on plant communities. The
present study is a step in that direction in that it demonstrates that CO2 can affect both ce11
elongation and stomatal density independent of any of its effects on photosynthesis. but.
clearly more work is required to elucidate the mechanism(s) by which CO2 modifies
developmental patterns.
-
References
B a n a r F.A. 1990. The response of natural ecosystems to the nsing global COz levels.
Annuai Review of Ecology and Systematics. 21: 167-1 96.
B a v a z F.A.. and Fajer. E.D. 1992. Plant life in a CO,-nch world. Scientific Amencan.
266: 68-74.
Beerling, D.J.. Chaloner. W.G, Huntley B. Pearson. J.A.. Tooley M.J and Woodward. F.I.
1992. Variations in the stomatal density of Salix herbacea L. under the changing
atmospherk CO, concentrations of late-and post-glacial time. Philosophical
Transactions of the Royal Society London. B 336: 21 5-224.
Camp, P. J and Wickliff, J.L. 1 98 1. Light or ethy lene treatments induce transverse ce11
enlargement in etiolated maize mesocotyis. Plant Physiology. 67: 125-1 28.
Ceulemans. R.. L-VanPraet and Jiang, X.N. 1995. Effects of CO, enrichment. leaf
position and clone on stomatal index and epidermal ceil density in poplar (Popzilzrs).
New Phytologist. 13 1 : 99- 107.
Cheverry. J.L.. Sy. M.O.. Pouliqueen. J. and Marcellin. P. 1988. Regulation by carbon
dioxide of 1 -arninocyclopropane- 1 -carboxylic acid conversion to ethylene in
climactric fmits. Physiologia Plantarum. 72: 535-540.
Eamus D. P.G. Jarvis. 1989. The direct effects of increase in the global atrnosphenc CO'
concentration on natural and commercial temperate trees and forests. Advances in
Ecologica~ Research. 19: 1-55.
Eisinger. CI W. 1983. Regulation of pea intemode expansion by ethylene. Annual Review of
Plant Physiology. 34: 225-240.
-
Farnsworth. E.J. Ellison. A.M. and Gong. W.K. 1996. Elevated COz alters anatomy.
physiology. growrh. and reproduction of red mangrove (Rhiqhora mangle L.).
Oecologia. 108: 599-609.
Gaba V. and Black. M. 1983. The convoi of ce11 growth by light. In Encyclopedia of
Plant Physiology . Vol. 16(A). Photomorphogenesis. Ediled by W .Jr. Shropshire and
H. Mohr. Spnnger-Verlag, Berlin. pp.358-400.
Gale. J. 1972. Availability of carbon dioxide for photosynthesis at high latitudes:
theoreticai considerations. Ecology. 53: 494-497.
Hicklenton. P.R. and Jolliffe. P.A. 1980. Carbon dioxide and flowering in Phurbitus ni1
Choisy. Plant Physiology. 66: 13- 17.
Hopkins. W.G. 1993. Introduction to Plant Physiology. John Wiley and Sons. INC.
Jarvis. P.G. 1987. Water and carbon Buses in ecosystems. Ecologicai studies. 61 : 50-67.
Jarvis. P.G. 1989. Atmospheric carbon dioxide and forests. Philosophical Transactions of
the Royal Society. B 321: 369-393.
Korner. C.H. Neurnayer M. Menendez-Riedl SP. Smeets-Scheel A. 1989. Functional
morphology of mountain plants. Flora. 182: 353-383.
Lauber. W. and Komer. C.H. 1997. In situ stomatal responses to long-term CO2
enrichment in calcareous grassland plants. Acta Oecologia. 18(3): 22 1-229.
Lu. W.P. and Kirkharn. M.B. 1992. Ethylene evolved by C' and C, grasses with twicr
carbon dioxide. Plant Physiology (supplement). 99: 147.
Madsen. E. 1973. Effects of CO, concentrations on the morphological. histological and
cytological changes in tomato plants. Acta Agriculturai Scandinavica. 23: 24 1-246.
-
Malone. SR.. Mayeux. H.S.. Johnson. H.B.. and Polley. H. W. 1993. Stomatal density
and aperture length in four plant species grown across a subambient CO, gradient.
Amencan Journal of Botany. 80: 141 3-14 18.
McElwain. J-C., Mitchell. F.J.C and Jones, M.B. 1995. Relationship of stomatal density
and index of Salk cinerea to atmosphenc carbon dioxide concentrations in the
Holocene. The Holocene. S(2): 2 16-2 19.
Mer. C.L. and Richards, F.J. 1950. Carbon dioxide and the extension growth of etiolated
oat seedlings. Nature. 165: 1 79- 1 80.
Morison. J.I.L. 1987. Plant growth and CO, history. Nature 327: 560.
O'Leary, J.W.. and G.N. Knecht. 198 1. Elevated CO, increases stomatal numbers in
Phaselotis vtrlguris leaves. Botanical Gazette. 142: 438-44 1.
Pearcy. R.W., and Bjorkman. 0. 1983. Physiological effects. - In: Lemon. E.R. (ed): CO,
and plants: The response of plants to rising levels of atmospheric curbon dio.ride.
Boulder. Colorado: Westview. 65- 105.
Pefiuelas. J. and Matamala. R. 1990. Changes in N and S leaf content. stomatal density
and specific leaf area of 14 plant species during the last three centuries. Journal of
Experimental Botany . 41 : 1 1 1 9- 1 124.
Poole. J.D.B.. B. Weyers. T. Lawson and J.A. Raven. 1996. Variations in stomatal
density and index implications for palaeoclimatic reconstructions. Plant Ce11 and
Environment. 19: 705-7 12.
Poorter. H. 1993. Interspecific variation in the growth response of plant to an elevated
arnbient carbon dioxide concentration. Vegetatio. 104/105: 77-97.
-
Purohit. A.N. and Tregunna. E.B. 1974. Effects of carbon dioxide on Pharbitus.
Xanfhizm. and Silene in short days. Canadian Journal of Botany. 52: 1283-1291.
Radoglou. KM.. and Jarvis. P.G. 1990. Effects of CO, enrichment on four poplar clones.
II. Leaf surface properties. Annals of Botany. 65: 627-632.
Radoglou. KM.. and Jarvis. P.G. 1992. Effects of CO, e ~ c h m e n t and nutrient supply on
grow-th and leaf anatomy of Phaseious vvulgaris L. seedlings. Annals of Botany. 70:
245-256.
Rayle. D.L. and Cleland. R.E. 1970. Enhancement of wall loosening and elongation by
acid solutions. Plant Physiology. 16: 250-253.
Reekie. E.G.. and Bazzaz. F.A. 1991. Phenology and growth in four annual species
grown in ambient and elevated COz. Canadian Journal of Botany. 69: 2475-248 1.
Reekie. J.Y.C.. Hicklenton. P.R. and Reekie. E.G. 1994. Effects of elevated CO, on time
of flowering in four short-day and four long-day species. Canadian Journal of
Botany. 72: 533-538.
Reekie. J.Y.C.. Hicklenton. P.R. and Reekie. E.G. 1997. The interactive effects of carbon
dioxide e ~ c h m e n t and daylength on growth and development in Prrrtnia hybrida
Annals of Botany 80: 57-64.
Rowland-Bamford, A.J., Nordenbrock, C.. Baker, J.T., Bowes. G. and Allen L.H.. JR.
1990. Changes in stomatal density of rice grown under various CO2 regimes with
natural solar irradiance. Environmental and Expenmental Botany. 2: 175- 180.
Ryle. G.J.A.. and J. Stanley. 1992. Effect of elevated COz on stomatal size and
distribution in peremial ryegrass. Annals of Botany. 69: 563-565.
-
Salisbury. E.J. 1917. On the causes and ecological significance of stomatal fiequency.
with special reference to woodland flora. Philosophical Transactions of the Royal
Society London. B216: 1-65.
Salisbury. F.B. and Ross. C. 1992. Plant physiology. Fourth edition. Belmont CA:
Wadsworth.
Sloane. M. and Sadava D. 1975. Auxin. carbon dioxide and hydrogen ion. Nature. 257:
68.
Strain. BR. and Cure. J.D. 1985. Direct effects of increasing carbon dioxide on
vegetation. Springfield. Virginia. USA: United States Department of Energy.
National Technical Information Service.
Stuiver. M. 1978. Atmosphenc carbon dioxide and carbon reservoir changes. Science.
199: 253-258.
Wittwer. S.H. 1992. Rising carbon dioxide is great for plants. The fall 1992 issue of
Policy Review to subscribe to Policy Review.
Woodwell, G.M. 1978. The carbon dioxide question. Scientific Arnerïcan. 238: 34-43.
Woodward. F.I. 1 986. Ecophysiological studies on the shrub C'acciniurn ~vrri l lus L.
taken from a wide altinidinal range. Oecologia. Berlin. 70: 580486.
Woodward. F.I. 1987. Stomatal numbers are sensitive to increase in CO1 fiom pre-
industrial levels. Nature. 327: 6 1 7-6 18.
Woodward. F.1, and Bazzaz. F.A. 1988. The responses of stomatal density to CO, partial
pressure. Journal of Espenmental Botany. 39: 1 77 1 - 1 78 1.
Vanderhoef, L.N. and Dute. R.R. 198 1. Auxin-regulated wall loosening and sustained
growth in elongation. Plant Physiology. 67: 146- 149.
-
Zebian, K.J and Reekie, E.G. 1998. The interactive ef5ects of atrnosphenc carbon dioxide
and light on stem elongation in seedlings of four species. Annals of Botany. 81:
185-193.
-
Appendix 1
Solutions for SEM use
Solution A and solution B
Solution A
NaZHP0,.2H,0 35.61g
Na2HP0,. 7H20 53.65g
Make up to lOOOml with distilled water
Solution B
NaH,PO,. H,O
Make up to lOOOml with distilled water
0.2M PO, buffer
Mix 22ml solution A and 77ml solution B to make 0.2M buffer.
Buffer may be stored in the fndge for up to one month.
0.1M PO, Buffer
Dilute 0.2M buffer with an equal amount of distilled water
2.5% Glutaraldehyde Solution-for fixation
0.2M PO, buffer 50ml
25% glutaraldehyde 1 Oml
Make up to 1 OOml with distilled water.
1 O h osmium tetroxide (OsO,) fixative
-
4% oso,
0.2M PO, buffer
5.4% sucrose
Distilled water
Store in brown glass bottle.
Discard when solution begins to discolor.
5m1
1 OmI
2m1
3m1
-
Sample preparation for SEM
1. Chernical fixation:
2.5% Glutaraldehyde buffer for 2-4 hours.
Wash three successive washes with phosphate buffer (0.1 M).
Postfix for I h in 1 % buffered OsO,.
Wash twice with O. 1 M phosphate buffer.
2. Dehydration:
Acetone dehydration in graded senes as follows for 10 minutes each-15. 30. 50. 70.
80.90. and 95%.
Absolute acetone: three changes for 10 minutes each.
3. Drying:
Critical Point Dry (CPD) tissues using acetone as the transitional tluid and liquid
COz.
1. Mounting specimen:
Mount cotyledons on specimen stubs and coat with 15-20nm gold-palladium
(60:40w/w).
-
Apaendix 3
Paraff~n Embedding Technique For Light Microscop~
Preparation of materia1:Cut the stem O.km below the cotyledons into sections not
longer than O.5cm long.
Fixation: FAA fixed tissues will be used (material may be left in FAA (90 ml 70%
alcohol: 5rnl glacial acetic acid; 5ml formalin) almost indefinitely).
Tertiary Butvl Alcohol (TBA) dehvdration schedule and infiltration:
TBA solutions are prepared as follows:
Dist. Water 95% Alcohol TBA 100% Alcohol
TBA 1 (50%) 50 ml 40 ml 10 ml
TBA 2 (70%) 30 ml 50 mI 20 ml
TBA 3 (85%) 15 ml 50 ml 35 ml
TBA 4 (95%) 45 ml 55 ml
TBA 5 ( 100%) 75 ml 25 ml
Note: A small amount of stain (such as saEranin) is added to TBA 5 in order to be able
to see extremely white or transparent material.
Keep material in a closed vial. Make the necessary changes by decanting off the old
solution and adding the new.
-
Start with matenal in 70% ETOH (30 min).
Transkr to 95% ETOH (30 min).
100% ETOH (30 min).
Samples are processed through the following stages: -
1. TBA 1 2 hours
2. TBA 2 2 hours
3. TBA3 2 hours
4. TBA4 2 hours
5. TBAS 2 hours ( c m be lefi overnight)
6. Absolute TBA* 4 hours
7. Absolute TBA 4 hours
8. Absolute TBA** overnight
9. Add liquid Tissue Mat (Le. paraffin) to clean vials and let harden.
Place tissue (with enough absolute TBA to just cover the tissue) on the
hardened Tissue Mat. Cork (loosely) and place in the oven until the Tissue
Mat has rnelted. (Takes approximately 24 hours).
10. Remove corks fiom vials and leave them in oven until al1 the TBA has
evaporated. This takes fiom 1-2 days at the end of which time a slight odor
is given off tiom the vials.
11. Decant and add Tissue Mat (2 changes in one day).
* Place on top of oven since absolute TBA solidifies at room temperature.
** If material is to be stored at this stage place it in a solution of 50°/o paraffin
-
oil and 50% TBA to prevent it fiom hardening.
12. Embed materiai. Matenai is ready to be mounted. trirnmed and sectioned
on a rotary microtome.
Steps no. 1-9: Material should be in corked vials
Steps no. 1 O- I l : Matenal should be in open vials.
D. Embeddin~ Material:
Embedding "boats"
Tweezers
Needles or probes
Aicohol larnp or Bunsen bumer
Pour a small amount of Tissue Mat into the embedding boat and allow it to harden
slightly, then fil1 the boat to about % full. Place it on the w r m part of the hot plate. Using
a heated tweezer remove the material from the via1 and place it into the embedding boat.
Reposition the material if needed with a hot needle.
Note: In order to work with hot parfiin. the above technique should be done quickly
and carefully. Once the material is properly positioned. very carehlly move the
embedding boat ont0 the cool portion of the warming table.
E. Trimmin~ and mountine paraffin blocks:
Trim the parafin blocks with a sharp single edged razor blade.
Leave about a 2 mm border of wax around the embedded tissue.
-
Mount the tnmmed block ont0 the wooden blocks using a heated spatula.
Keep the blocks in the Fndge overnight.
F. Sectioniw and mountiw ribbons onto elass slides:
In order to mount the tissue sections. the following supplies will be needed:
Distilled water
Al burnin
Glass slides
Needle or probe
Camel-hair brush
Kimwipes
Black p e n d
Put one drop of albumin on the slide and spread it into an even. thin coating using
Kirnwipes.
Cut ribbon into lengths (approximately 3 cm) and arrange on slide using a needlr or
brush. making sure that you maintain a serial order going from left to right. Put enough
water on the slide to float the sections. Leave slides to dry in 37C oven till become
completely dried.
-
1. Dewax tissue: Remove paraffin as follows:
100% xylene-5 min.
100% xylene-5 min.
100% EtOH-5 min.
2. Rehvdration: rehvdrate tissue as follows:
95% EtOH-5 min.
70% EtOH-5 min.
50% EtOH-5 min.
30% EtOH-5 min.
Distilled H,O-5 min.
3 . Stainin~:
1 O h Safiinin in distilled H,O-5 to 10 min.
Rinse in tap HIO-5 min.
4. Dehvdration:
30% EtOH-1 min.
-
50% EtOH- 1 min.
70% EtOH- 1 min.
95% EîOH-1 min.
5 .
0.5% Fast Green in 95% EtOH-1 to 2 min.
100% EtOH-1 min.
100% EtOH- 1 min.
100% EtOH-1 min,
6. Final removal of paramu:
1 0O0!0 xy lene-5 min.
100% xylene-5 min.
7. Mount in permount and apalv cover slip:
Use Acid ETOH to clean the cover slip and tlame it to dry. Put a permount on the
slide and cover with a cover slip.
-
l MAGE EVALUATION TEST TARGET (QA-3)
APPLlED - INIAGE . lnc = 1653 East Main Street - -. - - Rochester. NY 14609 USA -- -- - - Phone: 71 6/42-0300 -- -- - - Fa: 71 61288-5989 O 1993. Appiied Image. Inc.. Ail Fùghts R e s e W