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

List of Tables

Table 1. The level of significance for the effect of carbon dioxide on

matornical and morphological characteristics of seven species

.... ... ................................................................ 25

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

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

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

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

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.

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.

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

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.

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

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

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

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

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 .

(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.

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

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

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

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.

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

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.

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.

Medicago

CO2 level (ppm)

Fig. 2. The effect of CO2 on stem height of six genera.

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.

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.

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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