ABSTRACT
EFFECTS OF OZONE AND WATER DEFICIT ON STOMATAL
KINETICS, DIEL TRENDS IN STOMATAL CONDUCTANCE,
GROWTH AND ALLOMETRY IN CROPS AND WEEDS
Tropospheric ozone (O3) is a stressor of natural and agricultural
ecosystems. Crop and weed species may respond differently to this stress. Effects
of O3 levels and water deficit (WD) on stomatal kinetics, nocturnal stomatal
conductance (gs), and biomass productivity were tested in different crops and
weeds in three separate studies. In the first study, half response time of stomata
was determined under five different concentrations of O3 ( 0 , 50, 100, 125 and 150
ppb) in a custom-designed gas exchange system using Pima cotton (Gossypium
barbadense L) and sorghum (Sorghum bicolor L.). The kinetics of both stomatal
closure and opening were slowed in a dose-dependent manner in cotton but not in
sorghum. In the second study, the effect of O3 and WD on diurnal and nocturnal
gs, leaf pigmentation, shoot and root productivity were evaluated on Pima cotton
under different levels of O3 (4, 59 and 114 ppb, 12 hr mean) and two levels of
water , well watered and water deficit (WD). Chronic O3 and WD reduced
daytime gs while high O3 increased nocturnal gs. Elevated O3 reduced the aboveand below-ground productivity of cotton but the imposed level of WD had no
effect. A third study, evaluated the effect of O₃ and water levels on Palmer
amaranth (Amaranthus palmeri L.) and common waterhemp (A. rudis L). There
was no effect of O3 or imposed level of WD on these species. Therefore, biomass
productivity may be affected more in crops than in weeds under elevated O3.
Furthermore, the weed species had significant nocturnal gs which could reduce soil
moisture levels.
Rama Paudel
May 2015
EFFECTS OF OZONE AND WATER DEFICIT ON STOMATAL
KINETICS, DIEL TRENDS IN STOMATAL CONDUCTANCE,
GROWTH AND ALLOMETRY IN CROPS AND WEEDS
by
Rama Paudel
A thesis
submitted in partial
fulfillment of the requirements for the degree of
Master of Science in Plant Science
in the Jorden College of Agricultural Sciences and Technology
California State University, Fresno
May 2015
APPROVED
For the Department of Plant Science:
We, the undersigned, certify that the thesis of the following student
meets the required standards of scholarship, format, and style of the
university and the student's graduate degree program for the
awarding of the master's degree.
Rama Paudel
Thesis Author
Anil Shrestha (Chair)
Plant Science
John Bushoven
Plant Science
David A. Grantz
University of California, Riverside
For the University Graduate Committee:
Dean, Division of Graduate Studies
AUTHORIZATION FOR REPRODUCTION
OF MASTER’S THESIS
X
I grant permission for the reproduction of this thesis in part or in
its entirety without further authorization from me, on the
condition that the person or agency requesting reproduction
absorbs the cost and provides proper acknowledgment of
authorship.
Permission to reproduce this thesis in part or in its entirety must
be obtained from me.
Signature of thesis author:
ACKNOWLEDGMENTS
First of all, I am grateful to Dr. Anil Shrestha for the guidance and support
during my graduate studies. I would like to extend my gratitude to my committee
members Dr. John Bushoven and Dr. David Grantz for their guidance and support.
Thank you, Dr. Grantz for the opportunity to work in your lab. I am thankful to
Hai-Bang Vu and Nadia Juarez for their help during my experiments.
I would like to extend my sincere appreciation to Dr. Sharon Benes for all
her support and inspiration. The support and encouragement from Dr. Charles
Boyer and Dr. Sandra Witte is precious. I am really grateful for the Harvey
Scholarship/ Jordan Assistantship.
Marlene, I cannot explain in words how amazing a person you have been.
Thank you very much for always being there when I needed anything. I would like
to thank all the professors and the wonderful people in Plant Science Department
and in Kearney Agricultural Research and Extension Center for their time and
support. I am thankful to Dr. Matthew Fidelibus and his team for the opportunity
to experience vineyards of Central Valley and learn many research ideas.
I would like to dedicate my thesis to my mother Durga Kumari Sharma and
my twin sister Rakshmi Paudel. Their love and support made it possible. I would
like to acknowledge my younger sisters Manisha Paudel, Anisha Paudel, my father
Gyanendra Prasad Paudel and my maternal uncle Arjun Subedi. You all are an
indispensable part of my life.
Last but not least, it will not be complete without acknowledging Reeta
Shrestha. My abroad study would not be complete without the love, support and
care from her and all Nepalese families in Fresno. Thank you all.
TABLE OF CONTENTS
Page
LIST OF TABLES ................................................................................................. vii
LIST OF FIGURES ............................................................................................... viii
INTRODUCTION .................................................................................................... 1
LITERATURE REVIEW ......................................................................................... 7
Ozone ................................................................................................................ 7
Tropospheric Ozone and Its Effect on Plants.................................................... 9
Ozone Effect on Plant Cell and Tissue ........................................................... 10
Water Stress and Its Effect on Plants .............................................................. 14
OBJECTIVES......................................................................................................... 18
OBJECTIVE I: THE KINETICS OF STOMATAL REGULATION IN
COTTON AND SORGHUM TO O3 OVER TIME PERIODS .................. 19
Introduction ..................................................................................................... 19
Materials and Methods .................................................................................... 23
Results and Discussion.................................................................................... 28
Conclusion....................................................................................................... 35
OBJECTIVE II: THE EFFECT AND INTERACTION OF O3 EXPOSURE
TO MILD WATER DEFICIT STRESS IN GROWTH AND
PHYSIOLOGICAL PARAMETER IN PIMA COTTON .......................... 37
Introduction ..................................................................................................... 37
Materials and Methods .................................................................................... 39
Results and Discussion.................................................................................... 43
Conclusion....................................................................................................... 55
OBJECTIVE III: THE EFFECT OF O3 EXPOSURE AND MILD WATER
DEFICIT STRESS IN GROWTH AND PHYSIOLOGICAL
RESPONSES IN SELECTED AMARANTHUS SPECIES (PALMER
AMARANTH AND COMMON WATERHEMP) ..................................... 57
vi
Page
Introduction ..................................................................................................... 57
Materials and Methods .................................................................................... 62
Results and Discussion.................................................................................... 68
Conclusion....................................................................................................... 81
LIST OF TABLES
Page
Table 1. Impact of ozone on the half time (t1/2) for stomatal response and
resulting water consumption by Pima Cotton. ......................................... 33
Table 2. Impact of ozone and irrigation regimes on daytime and nocturnal
steady state stomatal conductance and resulting water consumption
by Pima Cotton......................................................................................... 46
Table 3. Impact of ozone and irrigation regime on leaf pigmentation and leaf
properties of Pima cotton. ........................................................................ 49
Table 4. Impact of ozone and irrigation regime on shoot properties of Pima
cotton. ....................................................................................................... 51
Table 5. Impact of ozone and irrigation regime on root properties of Pima
cotton. ....................................................................................................... 52
Table 6. Impact of ozone and irrigation regime on calculated water
consumption of common waterhemp and Palmer amaranth .................... 71
Table 7. Impact of ozone and irrigation regime on leaf properties of Common
waterhemp. ............................................................................................... 74
Table 8. Impact of ozone and irrigation regime on leaf properties of Palmer
amaranth. .................................................................................................. 75
Table 9. Impact of ozone and irrigation regime on shoot parameters of
Common waterhemp. ............................................................................... 77
Table 10. Impact of ozone and irrigation regime on shoot parameters of
Palmer amaranth. ..................................................................................... 78
Table 11. Impact of ozone and irrigation regime on root parameters of
Common waterhemp. ............................................................................... 79
Table 12. Impact of ozone and irrigation regime on root parameters of Palmer
amaranth. .................................................................................................. 80
LIST OF FIGURES
Page
Figure 1. Illustrative diagram of custom-designed gas exchange system ............. 26
Figure 2. Representative stomatal responses
to (a,c) a step increase in
-1
irradiance (100 to 1600 µmol m-2
s
), or (b,d) a step decrease in
irradiance (1600 to 100 µmol m-2 s-1) in Pima cotton during acute
exposure at 50 ppb O3 (a,b) or 100 ppb O3 (c,d). ................................... 29
Figure 3. Effect of acute O3 exposure in Pima cotton on the kinetics of
stomatal opening (circles; solid line) and stomatal closing (triangles;
dashed line) in-2 response
to a step change in irradiance from 100 to
1600 µmol m s-1 (opening) or 1600 to 100 µmol m-2 s-1 (closing). ...... 30
Figure 4. Simulated time courses of stomatal opening. ........................................ 32
Figure 5. Effect of acute O3 exposure in sorghum on the kinetics of stomatal
opening (circles; solid line) and stomatal closing (triangles; dashed
line) in response
to a step change in irradiance from 100 to 1600
µmol m-2 s-1 (opening) or 1600 to 100 µmol m-2 s-1 (closing). ............... 35
Figure 6. The effect of chronic ozone (O3) exposure on the diel timecourse of
stomatal conductance (gs) in Pima cotton (a) under well-watered
(WW) or b water-deficit (WD) conditions. Plants were exposed to
the low O3 treatment (LO3:4 ppb; 12 h mean) or the high O3
treatment (HO3;114ppb; 12 hr mean) mean ±S.E. .................................. 44
Figure 7. The effect of irrigation regime (averaged over chronic ozone (O3)
exposure) on the diel time course of stomatal conductance (gs) in (a)
Common waterhemp (b) Palmer amaranth. Plants were exposed to
well-watered (WW) and water deficit (WD) condition .......................... 70
Figure 8. The effect of irrigation regime and chronic ozone (O3) exposure on
Chlorophyll content (SPAD units) (a) Common waterhemp (b)
Palmer amaranth ..................................................................................... 73
1
INTRODUCTION
Climate change is a global phenomenon caused by increased emissions of
greenhouse gases. Several components of this phenomenon affect agroecosystems
in many ways resulting in different outcomes including shifts in productivity
(Fuhrer, 2003). Climate change depends on the combined effect of several factors
such as alterations in precipitation patterns, increases in temperature, and increase
in greenhouse gases including tropospheric ozone (O3). These factors affect plant
growth and development directly and can also affect crop productivity by altering
nutrient cycling patterns, soil water profiles, crop-weed interactions, and plant pest
occurrences (Fuhrer, 2003). Stressors such as water deficit (WD) and elevated O3
can act simultaneously on the plants (Herbinger et al., 2002) and can promote the
formation of harmful reactive oxygen species (ROS) that can cause chlorophyll
bleaching, protein oxidation, lipid peroxidation, and nucleic acid damages
(Herbinger et al., 2002).
Soil water deficit and tropospheric O3 are increasing in many agricultural
regions of the world as well as in unmanaged ecosystems (Field and Van Aalst,
2014, Grantz, 2003). The increase in O3 and WD conditions is driven by other
elements of global change such as increase in nitrous oxide (N2O) and methane
(CH4) (Field and Van Aalst, 2014, The Royal Society, 2008). Elevated O3 will
create novel and challenging conditions for production of food, fiber, and
bioenergy (The Royal Society., 2008, Stevenson et al., 2006, Vingarzan, 2004,
Wilkinson et al., 2012). Therefore, a better understanding of the characteristics of
plants and their responses to combination of stressors is needed to ensure efficient
crop production.
2
Ozone is a major oxidant air pollutant that is increasing globally with
population growth and industrialization (Grantz, 2003), and it is projected to
further increase in the future (Grantz et al., 2013). Tropospheric O₃ is phytotoxic
and is formed by photochemical reactions of CH₄ and other hydrocarbons in the
presence of NOx (NO + NO2) (Felzer et al., 2007). Several studies have reported
inhibitory effects of O₃ on photosynthesis, crop growth, and yield (Fiscus et al.,
2005). Ozone has been reported to cause yield losses ranging from 5 to 15% in
sensitive crop varieties (Booker et al., 2009). More than 20% of the crop
production land in Europe was estimated to have risk of yield losses of 5% or
more in 2002 (Mills et al., 2007). Modeled tropospheric O₃ concentration
combined with an experimentally-derived function indicated 10% yield loss in US
soybean (Glycine max L.) production by 2005 (Tong et al., 2007). In China,
simulation of cumulative O3 concentration suggested that soybean and wheat
(Triticum aestivum L.) yield were suppressed by 12% to 19% in 1990 (Wang and
Mauzerall, 2004). Different plant species or even plants within the same species of
different genotypes have shown differences in their sensitivity to O₃ (Dumont et
al., 2013).
Studies have also reported that O₃ can have differential effects on crops and
weeds and thereby alter crop-weed competition dynamics in some crops. Some
weeds such as black nightshade (Solanum nigrum L.), horseweed (Conyza
canadensis L. ) (Grantz et al., 2008), and yellow nutsedge (Cyperus esculentus L.)
(Shrestha and Grantz, 2005) have been found to be tolerant to O₃ and in some
cases more competitive with crops in elevated O₃ conditions.
Water deficit is another major factor limiting plant growth and productivity
around the world (Loka et al., 2011; Wani et al., 2009). Although advancements in
irrigation technology have improved crop productivity, irrigation costs and limited
3
water resources are restrictive factors in many parts of the world (Loka., 2011).
Availability and quality of water affect the growth and physiological processes of
all plants (Gardner and Gardner, 1983; Loka et al., 2011). Water plays a
predominant role in plant nutrient transport, chemical and enzymatic reactions,
cell expansion, and transportation (Kramer, 1980; Loka et al., 2011). Water stress
results in anatomical and morphological alteration as well as changes in
physiological and biochemical processes altering several plant functions (Kramer,
1980).
Water stress in plants depend both on supply of water to the soil and the
evaporative demand of the atmosphere (Loka et al., 2011). Generally, plant water
stress refers to the condition where the plant’s water potential and turgor are not
sufficient to perform normal plant function (Hsiao, 1973; Loka et al., 2011). Many
factors are responsible for the loss due to water stress such as the severity and
duration of the stress, the growth stage at which stress is imposed, the plant
species and their genotype (Kramer and Boyer, 1995), and competition from weed
species.
Although, O3 and WD interactions are common in the environment, their
effects on plant growth and development are poorly characterized (Fuhrer, 2003;
Hoshika et al.; 2013, Mansfield, 1973). Some studies have shown antagonistic
interactions between O3 and WD, where effects of O3 are reduced by increased
WD (Temple, 1986; Temple et al., 1988; Temple, 1990). This antagonistic effect
has been mainly attributed to changes in stomatal regulation. However, there are
some studies that have reported synergistic interactions between these two
variables (Heggestad and Lesser, 1990). Interactions of O3 and WD for biomass
productivity and morphological traits are not well understood.
4
Stomata are the major regulators for both carbon (C) and water cycles at
individual plant and regional scales (Massman et al., 2000). Therefore, an accurate
measurement of stomatal responses and their combined effect with C assimilation
is important for numerous applications. Some studies have shown decrease in
transpiration and increases in water runoff with elevated CO2 in temperate and
boreal forests (Gedney et al., 2006; Keenanet al., 2013). However, in the case of
O3, indirect evidences have suggested that elevated O3 increased transpiration and
reduced runoff and stream flow (McLaughlin et al., 2007; Sun et al., 2012;
Uddling et al., 2009). The reason behind these phenomena might be the impact of
O3 on nocturnal gs and dynamics of stomatal response (Uddling et al., 2009).
However, an accurate measurement of stomatal responses to O3 and their
combined result on C assimilation is not well described.
To estimate stomatal regulation of water use and ozone uptake, (Emberson
et al., 2000) used maximum assimilation (A) data and an idealized relationship
between A and gs. Ozone may uncouples the relationship between A and gs and
affects physiologically-based modeling of water use by ecosystems. Further,
incomplete stomatal closure occurs with moderate and higher O3 exposures
particularly at night. Recently, incomplete stomatal closure has been reported with
elevated O3 exposure during daytime in many species (Hoshika et al., 2013;
Hoshika et al., 2012; Mansfield, 1973). These effects will impact water use and
landscape-scale hydrology.
Both chronic and acute exposure to O3 have been shown in a variety of
systems to have three distinct impacts on stomatal regulation: reduced daytime
(diurnal) gs, slower (sluggish) stomatal responses to environmental stimuli, and
increased nighttime gs (incomplete nocturnal closure) (Grams et al., 1999; Grantz
and Yang, 1996; Grulke et al., 2007a; Hoshika et al., 2013; Kellomaki and Wang,
5
1997; Kitao et al., 2009; Mansfield, 1973; Oksanen, 2003; Paoletti, 2005; Reiling
and Davison, 1995). Moreover, WD also has similar impacts but O3 and WD
interactions are not well understood (Fuhrer, 2003; Grantz and Yang, 1996;
Hoshika et al., 2013; Mansfield, 1973). In addition, the relationships between
impacts of O3 and WD on stomatal behavior during different times of the day are
not fully understood and the impacts of these stressors on productivity, biomass
allocation, and functional parameters such as maintenance of plant water relations
have not been characterized. Generally, stomatal measurements are made at
midday when gas exchange is maximum. However, it is still not clear if the
midday gas exchange fully represents the response of stomata at other times of the
day or their impacts on plant function. Stomatal regulation may cause alterations
in avoidance of high evaporative demand and elevate O3 which contribute for
stress tolerance (Dumont et al., 2013).
In agroecosystems weeds are generally present with crops in the same plots.
Not much is known on the effect of O3 and WD or the interaction of these two
factors on the physiology of weed species. The factors discussed above on gs and
stomatal regulation can very likely affect weed species; however, the level of
influence may be less in weed species than in crops as weeds generally respond
and adapt to changing environmental conditions better than crops and this can
have ecological and agronomical implications (Peters et al., 2014). Hence, from an
agroecosystem perspective, it is also important to assess the influence of O3 and
WD on some common weed species.
Therefore, in this study, three different experiments were conducted to
examine the effect of O₃ on stomatal dynamics of plants with different
physiognomic classes. The studies tried to assess the response of stomatal kinetics
to acute exposure to O₃ over time periods of minutes to hours, and evaluate the
6
effect of chronic exposure to O₃ on day and night time gs. This research also
attempts to address inaccuracies in projecting effects of increased O₃ on landscape
hydrology and crop water use due to inadequate physiological characterization of
stomatal response to environmental parameters in the atmosphere. Additionally,
the study also attempts to identify the potential challenges in crop-weed
competition in agroecosytems under elevated O₃ and increasing water scarcity
using cotton (Gossypium sp.) as a model crop and two common Amaranthus weed
species.
7
LITERATURE REVIEW
Ozone
Ozone (O3), a gaseous atmospheric constituent, is a triatomic form of
oxygen (O2). In the stratosphere, O3 and hydroxyl radical (OH-) is formed from
O2 through a series of reaction driven by sunlight (Schlesinger and Bernhardt,
2013). Stratospheric O3 protects the earth’s biosphere from harmful ultraviolet
radiation (Hocking et al., 2007) whereas; in the troposphere O3 is a harmful byproduct of anthropogenic pollution. However, some of the O3 produced in the
stratosphere is transported to the troposphere by stratospheric and tropospheric air
exchange (Hocking et al., 2007; Schlesinger and Bernhardt, 2013). Ozone and OH
are primary gases that can oxidize many trace gases to CO2, nitric acid (HNO3),
and sulfuric acid (H2SO4) (Schlesinger and Bernhardt, 2013).
When nitrogen dioxide (NO2) is present in the atmosphere, it is dissociated
by sunlight,
NO2 + hv
NO + O,
Where hv is ultraviolet light with wavelength < 318 nm and O is an excited
atom of oxygen.
The atomic oxygen produced in the reaction above is combined with
oxygen and produces ozone:
O + O2
O3
Thus, the net reaction is,
NO2 + O2
NO + O3
This is an equilibrium reaction, so high concentrations of NO tend to drive
the reaction to form nitrogen dioxide. Since sunlight is essential to form O3 by
these pathways, therefore, these are known as photochemical reactions
8
(Schlesinger and Bernhardt, 2013). At night, O3 is consumed by reactions with NO
to form NO2, some of which may go further to HNO3 (Schlesinger and Bernhardt,
2013).
Nitrogen dioxide and nitric oxide (NO) which are collectively known as
NOx are found in polluted air derived from automobile and industrial emissions
(Felzer et al., 2007). NOx can be found in small concentrations in the natural
atmosphere produced from forest fire, lightening discharges, and microbial
process in the soil (Schlesinger and Bernhardt, 2013). Therefore, formation of O3
from NO2 is a natural process. However, industrial and automobile emissions have
simply raised the concentration of NO2 and other precursors to O3 formation
(Cooper et al., 2010; Lelieveld et al., 2004; Schlesinger and Bernhardt, 2013).
Ozone is subject to further photochemical reactions in the troposphere,
O3 + hv
O2 + O
Reaction of O with water yields hydroxyl (OH) radicals:
O + H2O
2OH.
Some O3 in troposphere also comes from stratosphere by mixing with
stratospheric and tropospheric air (Schlesinger and Bernhardt, 2013).
O3 + OH
HO2 + O3
HO2 + O2
OH + 2O2
In polluted atmospheres, this OH radical oxidizes carbon monoxide (CO).
CO + 2O2 + 2OH
CO2 + O3 + H2O
Similarly, the oxidation of (CH4 in the presence of high concentration of
NO proceeds through a large number of steps, yielding a net reaction of
CH4 + 4O2
CH2O + H2O + 2O3
Proper understanding of the changes in the OH and other oxidizing species
in the atmosphere is important to predict future trends in the concentration of trace
9
gases such as CH4 which can contribute to global warming (Schlesinger and
Bernhardt, 2013).
Tropospheric Ozone and Its Effect on Plants
Tropospheric O3 is a major oxidant air pollutant that is increasing globally
(Grantz, 2003). Although the pattern of O3 is not consistent in all locations, current
level of O3 in many areas causes injury to agro ecosystems and unmanaged
ecosystems (Ashmore, 2005; Booker et al., 2009). Many studies have reported
inhibitory effects of O₃ on plants since four decades (Fiscus et al., 2005).
The level of O₃ sensitivity is dependent on the defense mechanism of plants
which is influenced by the regulation of gs and detoxification of reactive O2
species generated during O₃ degradation (Dumont et al., 2013). Both chronic and
acute exposure to O3 have been shown to cause three distinct impacts on stomatal
regulation such as reduced daytime (diurnal) gs, slower (sluggish) stomatal
responses to environmental stimuli, and increased nighttime gs (incomplete
nocturnal closure) in a variety of systems (Grams et al., 1999; Grantz and Yang,
1996; Grulke et al., 2007b; Hoshika et al., 2013; Kellomaki and Wang, 1997;
Kitao et al., 2009; Mansfield, 1973; Oksanen, 2003; Paoletti, 2005; Reiling and
Davison, 1995). Water deficit has similar impacts, though O3 and water
interactions are not well understood (Fuhrer and Booker, 2003; Grantz and Yang,
1996; Hoshika et al., 2013; Mansfield, 1973).
Current and anticipated O3 levels can cause direct damage to vegetation
(Booker, Muntifering, et al., 2009), suppress agricultural yield (Avnery et al.,
2011; Emberson et al., 2009; Heck et al., 1984) and inhibit growth of native
vegetation (Materna, 1984), alter plant growth and structure (Heagle et al., 1988b),
and impact daily maximum gs and photosynthesis (Reich and Lassoie, 1984) thus
10
curtailing their duration and maximal rates (Paoletti and Grulke, 2005). Plant WD
has similar impacts as that of O3, as both reduce the magnitude and duration of
daytime gas exchange and suppress biomass production (Grantz and Shrestha,
2006; Grantz and Yang, 1996; Heagle et al., 1988a; Tingey et al., 1971).
Ozone Effect on Plant Cell and Tissue
Ozone movement from the surface into the interior of the plant cell and the
subsequent cell reactions depends on the dose of O3 and the protector system of
the plant cell (Roshchina and Roshchina, 2003). The initial barrier to O3 is
comprised of excretions and cellular antioxidants (Fiscus et al., 2005; Roshchina
and Roshchina, 2003). Once O3 reaches the plasma membrane, it interacts with the
sensory and antioxidant components of the plasmalemma (Roshchina and
Roshchina, 2003). At high concentrations, O3 passes through the plasmalemma
and can come into direct contact with the cytoplasm and cellular organelles
(Heath, 1994; Roshchina and Roshchina, 2003).
There are both direct and indirect mechanisms of O3 action in the plant
cells. Ozone interacts directly with cellular components at small concentration for
surface ingredients of the cell wall, excretions and plasmalemma such as lipids,
proteins, phenols and terpeniods etc. (Fiscus et al., 2005; Heath, 1994; Roshchina
and Roshchina, 2003). If the concentration of O3 is high, some oxidizing potential
or ROS enters the cell by damaging plasma membrane and affects the intracellular
ingredients including nucleic acids (Roshchina and Roshchina, 2003). Indirect
action of O3 especially consists of ozonolysis of cellular component or the water in
the cell which results in the formation of some active products such as free
radicals, OH-and H2O2 (Fiscus et al., 2005; Roshchina and Roshchina, 2003). Such
products can act as chemosignals outside of plasmalemma and induce a cascade of
11
secondary messenger reactions within the cells (Roshchina and Roshchina, 2003).
If the concentrations of O3 derivatives are high it can also damage cellular
components (Roshchina and Roshchina, 2003).
The basic pathway that O3 penetrates into the plant tissue is through the
stomatal openings (Hoshika et al., 2013). Ozone enters into the leaf through the
stomata by diffusion based on the concentration gradient (Roshchina and
Roshchina, 2003). Then it flows through the intercellular spaces into the
parenchyma cells. Therefore, factors reducing stomatal conductivity would also
reduce the negative effects of O3 (Roshchina and Roshchina, 2003). Many studies
have observed a sharp decline in stomatal conductivity in plants upon fumigation
with O3 (Temple, 1986, 1990). However, this depends on a number of factors such
as O3 concentration in the atmosphere, exposure time, and the characteristics of
plant species. Under high concentrations of O3, the stomata closing results in a
reduction in O3 absorption (Guderian, 1985; Roshchina and Roshchina, 2003).
While studies have reported that O3 damage levels correlate with the size of
stomata apertures, which increases with an increase in humidity (Otto and Daines,
1969). On the contrary, a study reported that an increase in abscisic acid induced
stomata closure and reduced O3 damage (Roshchina and Roshchina, 2003). Ozone
impedes the function of guard cells. The level of this influence on stomatal
conductivity depends on many factors including plant age and level of light
exposure. Further, it has been reported that young leaves have higher stomatal
conductivity than old ones (Reich, 1987; Roshchina and Roshchina, 2003).
Ozone usually penetrates through the stomata and enters into the leaf
(Grulke et al., 2004). During this course, it passes through air-filled portions of
intercellular spaces and is absorbed by the damp cell walls of the mesophyll
(Roshchina and Roshchina, 2003). A number of reactions may occur along the
12
route resulting in decomposition of O3 in the water phase of cellular medium, such
as the reaction of O3 with antioxidants and olefins excreted by plant cells
(Roshchina and Roshchina, 2003). During the interaction with water, O3
decomposes into radicals and peroxides (Gurol and Singer, 1982; Roshchina and
Roshchina, 2003).
In the case of leaf cell walls and cavities, the entry of O3 is carried out
through the plant produced olefins, mainly ethylene and isoprene (Roshchina and
Roshchina, 2003). In a layer of a liquid surrounding the mesophyll cells of plants,
concentrations of OH- radicals and peroxides are increased at the expense of the O3
concentration (Roshchina and Roshchina, 2003). The O3 concentration in the
intercellular spaces as well as cellular environment of plant leaves may decrease
due to reactions with endogenous hydrocarbons. However, Chameides (1989)
suggested that the reactions of O3 with the water contained in the call wall and
olefins are not important for O3 absorption.
Some of the O3 that diffuses into a leaf reacts with the ascorbic acid
contained in the cell wall. According to Chameides (1989) the plants can protect
themselves by concentrating ascorbate in the cell wall. Ascorbic acid reacts with
O3 and prevents the penetration of O3 inside the cell wall and thus stops O3 from
reaching the more vulnerable part of the cell located inside the plasmalemma
(Roshchina and Roshchina, 2003). Besides ascorbic acid, other antioxidants in
apoplast such as phenols, amino acids containing sulfhydryl group and other
compounds also prevent the penetration of O3 inside the cell (Roshchina and
Roshchina, 2003). Redox enzymes (superoxide dismutase, peroxidase and
catalase) can also execute the detoxification of O3 in plant cells (Roshchina and
Roshchina, 2003). They protect living cells against free radicals and peroxides
formed in reactions between O3 and unsaturated hydrocarbon (Roshchina and
13
Roshchina, 2003). Thus it is likely that detoxification of O3 in the apoplast is one
of the primary means employed by cells to protect themselves against O3
(Roshchina and Roshchina, 2003).
Chronic ozonation of plants causes adaptive or damaging changes in plant
metabolism (Roshchina and Roshchina, 2003). The stable quantity of antioxidant
in plant cells undergoes an increase and new antioxidants or other protective
compounds are formed. The toxic damage inflicted by O3 on the cells are similar
to the damages caused by several other factors which are first displayed at the
biochemical level before showing visible morphological symptoms (Duccer and
Ting, 1970; Roshchina and Roshchina, 2003). Photosynthesis, respiration,
nitrogen, and lipid exchanges are all affected along with the activities of the
secondary metabolism (Roshchina and Roshchina, 2003). Later visible damage
such as necrotic spots and bronze coloration on leaves appear (Fiscus et al., 2005;
Heath, 1994; Roshchina and Roshchina, 2003).
Ozone exposure changes the carbohydrate metabolism in plant cells. The
effects are attributed to changes in the processes of photosynthesis and respiration
as a whole, in the glycolytic and the pentose phosphate pathways, as well as in the
synthesis and catabolism of starch and cellulose. The effects of O3 on
photosynthesis are different for different plant species (Guderian, 1985; Roshchina
and Roshchina, 2003). They are mostly dependent on the dose of O3. High doses
of O3 are required for inhibition of photosynthesis in higher plants (Bennett and
Hill, 1974; Ormrod et al., 1981). The main reason for photosynthesis inhibition in
plants is a decrease in carboxylation intensity. Ozone is known to be responsible
for the premature decline in the activity and quantity of rubisco (Eckardt and Pell,
1994). The premature decline is responsible for the reduction in photosynthetic
14
capacity and can also induce acceleration of foliar senescence (Eckardt and Pell,
1994; Reich, 1983).
The O3-induced granulation in the stroma of chloroplast is believed to result
from the oxidation of the SH- groups in ribulose bisphosphate carboxylase (RubPcarboxylase) and changes in ion content in stroma (Dominy and Heath, 1985;
Mudd, 1981; Roshchina and Roshchina, 2003). The inhibition of the enzyme
occurs soon after exposure to O3. The RDP–carboxylase is a protein consisting of
large and small subunits organized in a uniform structure (Goodwin and Mercer,
1983). Landry and Pell (1993) indicated that fumigation with O3 causes the
enzymes to lose its large subunits, a factor that is perhaps the cause of its
deactivation, as the enzyme’s catalytic activity is connected with its large subunit.
Ozone can also have detrimental effects on the electron transport in both PSII
functions as well as in the xanthophyll cycle components (Fiscus et al., 2005).
At the tissue level, O3 effects are characterized as either acute or chronic
responses depending upon the type and appearance of visible symptoms (Fiscus et
al., 2005). Symptoms such as unregulated cell death and programmed cell death
are usually considered acute responses in which lesions occur within hours after
exposure to relatively high O3 concentrations (typically >150nmol mol-1) (Fiscus
et al., 2005). In contrast, the lesions which develop with exposure to lower
concentrations of O3 over days to weeks are included as chronic responses (Fiscus
et al., 2005).
Water Stress and Its Effect on Plants
Water is a major constituent of plants which comprises 80-90% of the
biomass of non-woody plants. Water is the central molecule in all physiological
process of the plants and is the major medium for transporting metabolites and
15
nutrients (Lisar et al., 2012). Water deficiency is one of the most important
environmental factor inhibiting photosynthesis, growth, and crop production under
field conditions (Boyer and McPherson, 1975; Chaves et al., 2009; Kramer, 1980).
Declining water supplies including ground water is creating deficiencies in many
parts of the world (Chaves and Oliveira, 2004). Approximately one third of the
cultivated area of the world suffers from inadequate water supplies (Basal et al.,
2009). Crop productivity in rain-fed agriculture is limited by water deficiency and
the severity of this problem is worsening with the increasing trend of climatic
uncertainties (Le Houerou, 1996).
Water deficiency is the situation where plant water potential and turgor is
not sufficient for a plant to perform its normal physiological functions (Lisar et al.,
2012). Water deficiency during critical growth and developmental stages
significantly reduces biomass accumulation, grain set, and grain yield and quality
in cereal crops (Agboma et al., 1997). In some areas of the world, many
horticultural crops such as grapevines depend upon irrigation water during water
deficit periods because rainfall occurs during the dormant season (De Souza et al.,
2005). Water deficiency reduces shoot growth which disrupts the flow of
carbohydrate form the source leaves to the developing sinks (Burke, 2007).
Water deficit condition causes a complex set of responses beginning with
stress perception which initiates a signal transduction pathway and shows changes
at cellular, physiological, and developmental levels (Bray, 1993). Under prolonged
water stress condition, plants dehydrate and die (Lisar et al., 2012). Low water
potential and turgor increases the solute’s concentration in the cytosyol and
extracellular matrices resulting in reductions in cell enlargement and growth
inhibition and failure in reproductive functions (Lisar et al., 2012). Water stress
causes closure of stomata and inhibition of photosynthesis (Bjorkman and Powles,
16
1984). Limitation of photosynthesis has been reported due to decreased rubisco
activity (Chaves and Oliveira, 2004). Rubisco is the key enzyme for carbon
metabolism in leaves and it acts as carboxylase in the Calvin Cycle and as an
oxygenase in photorespiration (Lisar et al., 2012). In addition, water stress
conditions acidify the chloroplast stroma inhibiting rubisco activity (Lisar et al.,
2012).
Water stress conditions also disrupt the cyclic and non-cyclic electron
transport during light reaction of photosynthesis (Lisar et al., 2012), and also
generate reactive oxygen species (ROS) such as superoxide, hydrogen peroxide
(H2O2) etc. (Chaves and Oliveira, 2004). Other effects of water stress include
reduced chlorophyll content in the leaves (Lisar et al., 2012), and reduced whole
plant leaf area because of a decrease in the initiation of new leaves or lack of
increase in leaf size or leaf abscission (Basal et al., 2009). Some plants are able to
adapt to water deficiency by shortening their growth cycle or by increasing root
growth thereby increasing water uptake (De Souza et al., 2005). Some plants cope
with water deficiency by either completing their active growth cycle during the
period when water is not limiting or by developing different responses to counter
water stress (Agboma et al., 1997; Turner et al., 1984). For example, some
deciduous species avoid water stress partially by reducing their foliage area and
restricting their growth during favorable seasons (Ain-Lhout et al., 2001).
The responses of stomata to leaf water status and environment are
important in regulating transpiration and photosynthesis. The relationships
between stomatal resistance, leaf water potential, leaf temperature, and
environmental factors such as temperature, and humidity are particularly important
to plants growing in arid or semiarid conditions. Stomatal regulation, or
adjustments that facilitates CO2 diffusion while minimizing water loss, might
17
enhance drought tolerance of plants subjected to temporal or sustained water
deficits (Ackerson et al., 1977). The sensitivity of stomata to water deficits
decreases with increasing leaf age in cotton and in the case of cotton and sorghum
(Sorghum bicolor L.), stomata tend to be less responsive to water stress if the
plants have been previously subjected to mild water deficit. Stomata of field–
grown plants often respond to leaf water potential in an entirely different manner
when compared with similar plants established in growth chambers (Ackerson and
Krieg, 1977). Stomata normally adjust to water stress by closing in response to
declining leaf water potentials. A concomitant increase in the endogenous level of
ABA often occurs in water-stressed tissue, suggesting that hormonal modulation
of stomatal response to water stress may be an important regulatory mechanism in
the ability of plants to withstand temporal water deficit (Ackerson, 1980).
18
OBJECTIVES
The specific objectives of this study are to: i) study the kinetics of stomatal
regulation in cotton and sorghum to O3 over time periods of minutes to hours, as a
function of acute O3 exposure; ii) investigate the effect and interaction of O3
exposure to mild water deficit stress in growth and physiological parameter in
Pima cotton; iii) investigate the effect of O3 and mild water deficit stress in growth
and physiological parameter in selected Amaranthus species.
19
OBJECTIVE I: THE KINETICS OF STOMATAL REGULATION
IN COTTON AND SORGHUM TO O3 OVER TIME PERIODS
Introduction
Stomatal movements occur due to the regulation of guard cell turgor
through the control of osmolyte concentrations in their cytosol (Pandey et al.,
2007). The basic function of stomatal regulation is to maximize C uptake while
minimizing water loss (Grulke et al., 2004). Stomata generally react to the
changing environmental parameters with their opening or closing. Stomatal
conductance can be calculated as a function of species type, phenology, and
environmental conditions (photon flux density), temperature, and vapor pressure
difference (VPD) since these are the most important factors in determining
stomatal aperture (Emberson et al., 2000). Stomatal response to O₃ is a complex
mechanism, which vary among species, leaf and plant age, and are affected by
other environmental conditions (Paoletti, 2005). Ozone has been reported to have
different effects on stomatal movements, it not only modifies the opening and/or
closing speed of stomata directly but also affects the opening and/or closing in
response to variations in light intensity (Hoshika et al., 2012), VPD (Grulke et al.,
2007), soil moisture (Hayes et al., 2012), CO2 concentration (Onandia et al.,
2011), and ABA concentration (Dumont et al., 2013). A study showed that
exposure to increased O₃ significantly depressed gs (Kellomaki and Wang, 1997).
Generally, short term exposure to O₃ causes rapid reduction in stomatal aperture,
while long term exposure to O₃ causes sluggish response of stomata (Paoletti,
2005). Some studies explained that the ‘sluggish’ closure of stomata was due to
guard cell wall delignification, as found in ozonated Picea abies (Paoletti, 2005)
and was associated with the effect of light (Tjoelker et al., 1995).
20
Ozone exposure modified the dynamics of stomatal regulation (Reich and
Lassoie, 1984) in poplar (Populus deltoides x trichocarpa), Norway spruce (Picea
abies (L.) Karst), and white fir (Abies alba Miller) (Keller and Hasler, 1984). The
authors described ‘sluggish’ stomatal responses to O3 with change in light
intensity. Others suggested the sluggish response of stomata in response to VPD
after exposure to ozone in sugar maple(Acer saccharum Marsh) and Scots pine
(Pinus sylvestrus L.) (Kellomaki and Wang, 1997; Tjoelker et al., 1995).
‘Sluggish’ stomatal responses with O3 exposure uncouples the relationship
between A and gs (Paoletti and Grulke, 2005) that affects physiologically based
modeling of water use by ecosystems. Further, incomplete stomatal closure occurs
with moderate and higher O3 exposures (Wieser and Havranek, 1995), particularly
at night (Grulke et al., 2004). This may significantly increase both continued O3
flux into the leaf and hence damage and further inhibit C uptake and increase
consumptive water use. Altered stomatal response to changing environmental
conditions persists for almost two weeks following the end of O3 exposure
(Paoletti, 2005). These effects will unavoidably impact water use in agro- and
natural-ecosystems and landscape-scale hydrology.
Many approaches have been developed for assessing O₃ risk to plants and
plant damage. Among them, two major methods are: a) exposure-based, and b)
flux-based. The exposure method is based on accumulation of daytime O₃ in the
air concentration. However, O₃ concentration in the air may not always be
correlated to O₃ fluxes into the leaves (Kurpius et al., 2002). Therefore, this
approach may not account for the ecological and physiological control of gs for an
accurate assessment of effective amount of O₃ entering into the leaves and
oxidizing the apoplast. The other important O₃-risk assessment method is fluxbased metrics that is dependent on accumulated stomatal fluxes above a
21
phytotoxic threshold (Karlsson et al., 2004). Recent studies have suggested that
this approach is more reasonable than exposure-based approach as it accounts for
the effective amount of O₃ responsible for plant injuries after entering through the
stomata. According to Emberson et al. (2000), stomatal O₃ fluxes are products of
gs (modeled in a multiplicative algorithm which accounts for the maximum gs and
all the phenological and environmental parameters affecting it, multiplied by O₃
concentration at the top of the plant canopy).
Therefore, for the correct assessments of O₃ effect on plants, information
only on O₃ concentration distributions is not sufficient (Cieslik, 2009). Literature
reviewed by Heath et al. (2009) correctly suggested that a stomatal-driven fluxbased approach may establish a phytotoxic threshold of accumulated O₃ fluxes,
with emphasis on high gs. The stomatal flux is only a fraction of the total O₃ flux
between the atmosphere and earth’s surface, i.e., the amount of O₃ molecules (or
moles) which crosses the air/surface interface per unit area and time (Cieslik,
2009). Stomatal O₃ flux is the quantity of O₃ molecules transferred per unit time
and area from air to the plant tissues through the stomata (Cieslik, 2009). The
exchange of gases between the atmosphere and the phytosphere is governed by the
ambient O₃ concentration, the turbulent conductivity of the lower atmosphere, and
the sink properties of the plants and soil. The dynamics of ambient O₃
concentrations are inherently coupled with the meteorology that governs its
synthesis and the deposition through effects on plant physiology. A flux-based
metric may help to reconcile responses observed in different exposure systems.
However, seasonal-average or flux-based approaches do not capture O₃ exposure
dynamics on a daily basis and their relationship to plant growth stages with
differing sensitivities (Booker et al., 2009).
22
A current model by (Emberson et al., 2000) that uses maximum
assimilation (A) data and an idealized relationship between A and gs (Farquhar
and Sharkey, 1982) to estimate maximum stomatal regulation of water use. While
the model parameterizes gs as a function of photosynthetic photon flux density
(PPFD), temperature, VPD, soil moisture deficit, and O₃ flux using hourly average
O₃ concentrations, the basic assumption is incorrect i.e., that gs scales with A
under O₃ impacted conditions (Paoletti and Grulke, 2005). Some models explain
the dynamic aspects of stomatal response, e.g. to light (Grantz and Zeiger, 1986)
and to VPD (Assmann and Grantz, 1990) and are increasingly used to explain the
impacts of steady state responses. Effects on stomatal opening and closing kinetics
are closely related to each other (Paoletti and Grulke 2010) and the magnitude of
steady state gs and the kinetics of both opening and closing, to evaluate net effects
on simulated water use.
Therefore, it is important to test stomatal responses to O3 in crop species
that is commonly grown in the San Joaquin Valley (SJV) of California where O3
concentrations in summer is generally the highest in the nation (Cramer
etal.,2000). Cotton is a C₃ plant of the Malvaceae family commonly grown as a
fiber crop. In addition, cotton has important uses in food and feed industries as a
source of cottonseed oil and protein-rich meal for livestock. Globally, the U.S. is
the third largest producer of cotton and the fifth largest consumer of cotton
(International Cotton Advisory Committee, 2014). In 2012, U.S. produced
approximately 17.3 million bales of cotton from 12.3 million planted acres.
California is the third major cotton producing state in the U.S. and produced 1.2
million bales of cotton from 0.367 million acres in 2012 (National Cotton Council
of America, 2014). Sorghum is multipurpose C₄ crop with diverse types of
species. It is the fifth major cereal crop cultivated in 37.85 million acres
23
worldwide. It is cultivated in 93 different countries across the world (FAO
Statistics, 2014) and is a staple food of many poor families in Africa, Asia, and
Central America (Girijashankar and Swathisree, 2009). Sorghum is also a major
source of ethanol and fodder for animal (Girijashankar and Swathisree, 2009). In
recent years, the importance of sorghum is also increasing as a biofuel crop. An
analysis of the U.S. Environmental Protection Agency (EPA) showed that the use
of grain sorghum can meet the 20% gas emission reduction threshold stipulated by
the Energy Independence and Security Act (EISA) of 2007 for conventional
renewable fuel (EPA, 2012). Sorghum is also considered as a potential energy
crop in California (Energy Weekly News, 2011). Both cotton and sorghum are
commonly grown in the SJV and are species in different physiognomic classes.
Therefore, the objective of this study was to determine whether stomatal responses
to O3 in cotton (a dicotyledonous C3 crop) and sorghum (a monocotyledonous C4
crop) could be directly measured in a custom designed gas exchange system and if
so, whether the response of stomatal kinetics to pulse exposure to different
concentration of O3 could be shown analytically to account for increased
transpiration and reduced stream flow as inferred from indirect measurements in
trees.
Materials and Methods
Plant Material
Seeds of Pima cotton (cv. S-6) were obtained from foundation seed stocks.
The seeds of forage sorghum were obtained from Scott seed. Seeds were
germinated on a bench in the research greenhouse at the University of California,
Kearney Agricultural Center (Parlier, CA USA; 103masl; 36.598 ̊N, 119.503 W
̊ )
in moist commercial seedling mix (Earthgro Potting soil, Scotts Company,
24
Marysville, OH, USA) contained in plastic pots (870 ml; 110mm x 110 mm x 125
mm; Dillen Products, Middlefield, OH, USA), and thinned to a single uniform
plant per pot.
Four- to 6-wk old plants were transferred to the laboratory and a sector of
the youngest fully expanded leaf, not containing a major vein, was placed in a
custom gas exchange system (modified after the system described by Grulke et al.
(2007a,b).
System Description
Due to lack of a commercially available gas exchange system which could
maintain elevated O₃ in a cuvette during an experiment, a custom-designed gas
exchange system was used to deliver known concentrations of O₃ to the leaves and
concurrently measure the foliar H2O, O₃, and CO₂ fluxes (HOC flux system).
The custom-designed system that supplied elevated O₃ was described by
Grulke et al. (2007a,b). This system was further modified to accurately measure
the flux using O₃ monitors (Model 49C). The system is a UV photometric gas
analyzer equipped with one inlet for reference (air entering the cuvette) and one
outlet for sample air (air exiting the cuvette). Atmospheric air was pumped by the
pump inside the monitors and was used for gas exchange. The dry air was
modified with a dew point generator (Model 610, LiCor Instruments, Lincoln, NE,
USA) passed to a custom refrigerator with a large peltier cooling block. Different
concentration of O₃ was generated from dry O2 in a custom-made O₃ generator
consisting of a UV light absorbed in the stream of O2. The combined flow of O₃
and the humidified and cooled reference air was pumped through the O₃ analyzer.
Small custom cuvettes (i.d. 2 × 3 × 1 cm), an empty control, and a sample
cuvette containing a leaf, were made of Plexiglass (acrylic) and illuminated from
25
above (100 µmol m-2 s-1 or 1600 µmol m-2 s-1) using red and blue LEDs (LI-640018; LiCor Inc.; Lincoln, NE, USA). A closed-cell foam gasket was used with the
cuvette. The exhausted air from the sample and the reference O₃ monitors was
passed to the sample and reference side of the CO2 and H2O IRGAS respectively
of a closed LiCor model 6400 cuvette. The flows of water and CO2 in sample
IRGA and water and CO2 in reference IGRA were monitored, matched, and
stabilized. In the HOC system cuvette, the LiCor illuminator (mixture of red and
blue light provided by light emitting diodes) were used to drive gas exchange. The
cuvettes were connected with fine-wire thermocouple to measure leaf level
temperature. The setup of the system is shown in Fig. 1.
Step changes in illumination were administered using the control circuitry
of a LI-6400 console to control the LI-6400-18. The rest of the plant was
illuminated during the measurement using similar LEDs [EcoSmart ECS 38 V2;
300K, 24 W (120W equivalent)]. Plants were allowed to acclimate to this system
until steady state gs was achieved.
Air was drawn from an isolated crawl space through a large buffer volume,
to both control and sample cuvettes. Humidity of the airstream entering both two
cuvettes was controlled with a dew point generator (LI-610) to maintain a leaf to
air VPD of approximately 1.7 kPa. Leaf temperature was determined with a
contact thermocouple (Type T, 76 µm dia) inserted to the abaxial surface of the
leaf. Leaf temperature, generally about 35 ºC, was lagged by 30 s in calculation of
VPD and gs to allow for flow from cuvette to analyzers. Water vapor and CO2
were monitored at 30 s intervals, using the cuvette of a steady state gas exchange
system (LI-6400), in series with matched ultraviolet O3 monitors (Model 41C;
Thermo Fisher Scientific Inc.; Waltham, MA, USA). The air stream to both
cuvettes was ozonated using a UV lamp with adjustable opaque cover. The
26
Figure 1. Illustrative diagram of custom-designed gas exchange system
27
airstream was cooled slightly with Peltier modules to offset the heat of the UV
lamp prior to stream splitting to the two cuvettes.
Exposure to Ozone
Mature leaves of the greenhouse grown plants were exposed to five
different concentrations (0, 50, 100, 125, and 150 ppb) of O3 as pulses, only on the
day of the experiment. Stomatal response of the leaves to varying PPFD levels
(100 to 1600 and 1600 to 100 µmol m-² s-1) was measured using the IGRAs in the
LI 6400 in 30-second intervals. The time course change in gs was used to
determine the half-response time (t½) and percent overshoot of final steady state
level. The experiment was repeated to produce at least three curves for decreasing
and increasing light intensity for each concentration of O3.
The acute O3 exposure experiment was performed on individual plants at
different concentration of O3, with the same concentration of O3 per day. Half
times were extracted by fitting single exponential equations to pulse response data
(Sigma Plot V.11.0). Stomatal responses to step changes in irradiance were
described as the t½.
Stomatal responses to step changes in irradiance were described as the half
response time (t½ = ln(2)/λ).
For stomatal closing
gs(t) =a + (b) e-λt
Eq. 1a
and for stomatal opening
gs(t) = a – (b)(1- e-λt)
Eq. 1b
Where a and b are fitted parameters related to the initial and final
magnitude of gs, λ is the fitted time constant, and t is time after the step change.
28
Projected Water Use
Canopy transpiration in each treatment was simulated from single leaf
measurements as:
T = (VPD) (gs) (LAI)
Eq. 2
Where T is transpirational water loss per second per square meter of leaf,
VPD is leaf to air vapor pressure difference, gs is single leaf stomatal conductance,
and LAI is leaf are per unit ground area. VPD was fixed at 0.02 mol mol-1 at night
and 0.04 mol mol-1 at midday, and LAI was fixed at 4 m2 m-2, similar to values
observed in a cotton field in California (Grantz et al. 1993, 1997).
Experimental Design and Data
Analysis
The experiment was arranged as a completely randomized design, with
each leaf being considered as a replicate. Data were analyzed using general linear
model procedure (PROC GLM) of SAS (SAS Institute, v. 9.2.1). Half times were
extracted by fitting single exponential equations to pulse response data (Sigma
Plot; v. 11.0, Systat Software Inc.). When the effect of treatment was significant
by F test (P ≤ 0.05), mean separation was determined using Fisher’s Least
Significant Difference (LSD) test.
Results and Discussion
The cotton plants showed substantial sensitivity and clear measurement for
kinetics were obtained from this system. At steady state, gs responded vigorously
to step changes in irradiance from 100 to 1600 µmol m-2 s-1 to induce stomatal
opening and from 1600 to 100 µmol m-2 s-1 to induce stomatal closing. The time
course of both opening and closing of stomata were described by a single
exponential relationship. The t½ of the closing response was slightly longer
29
(slower response) than the t½ of the opening. Representative time courses of
stomatal response at 50ppb and 100 ppb of O3 are shown in Fig. 2.
(a)
0.3
(b)
50 ppb
t1/2 = 2.65 min
50 ppb
t1/2 = 1.63 min
2
2
r = .975
P < .001
r = .997
P < .001
-2
-1
Conductance, gs (mol m s )
0.2
0.1
0.0
(c)
(d)
100 ppb
t1/2 = 5.03 min
0.5
100 ppb
t1/2 = 4.24 min
2
2
r = .968
P < .001
r = .984
P < .001
0.4
0.3
0.2
0
10
20
30
0
10
20
30
Time after step change in PPFD (min)
Figure 2. Representative stomatal responses to (a,c) a step increase in irradiance
(100 to 1600 µmol m-2 s-1), or (b,d) a step decrease in irradiance (1600 to 100
µmol m-2 s-1) in Pima cotton during acute exposure at 50 ppb O3 (a,b) or 100 ppb
O3 (c,d).
Note: Values of t½ were extracted from fitted single exponential functions obtained as (a) gs(t) =
0.0274 + 0.1387(1 – e-0..2615x); (b) gs(t) = -0.0036 + 0.1307(e-0.4264x); (c) gs(t) = 0.2509 + 0.1571(1 e-0.1378x)and (d) gs(t) = 0.2554 + 0.2199(e-0.1633x). Curves were fitted to the open circles, beginning
immediately after the step change in irradiance and ending when the new steady state was
attained. Solid circles represent the steady state before and after the response.
In O3-free air, the t½ of the opening response was approximately 2 min
(Fig. 3). The t½ of the closing response was generally more rapid than the t½ of
opening at the same concentration of O3 (Figs. 2, 3). The t½ for both opening and
30
closing increased linearly with O3 between 0 and 125 ppb (Fig. 3), though closing
was less sensitive to O3 than opening. At 125 ppb the t½ of opening increased by
3-fold and for closing by 1.7-fold, relative to no O3 exposure. The t½ for both
opening and closing increased by approximately 1.5-fold in current ambient O3
(about 40 ppb) relative to no O3 showing substantial slowing of stomatal response
(Sluggish response of stomata).
Figure 3. Effect of acute O3 exposure in Pima cotton on the kinetics of stomatal
opening (circles; solid line) and stomatal closing (triangles; dashed line) in
response to a step change in irradiance from 100 to 1600 µmol m-2 s-1 (opening) or
1600 to 100 µmol m-2 s-1 (closing).
Note: Means ± S.E. Linear regressions are fitted for each relationship separately, for data between
0 ppb and 125 ppb as (opening) gs = 0.0347 O3 +2.0883; (Closing) gs = 0.0148 O3 + 2.0814
The linear relationship broke down above 125 ppb O3, as both opening and
closing responses exhibited sharp declines in t½ as O3 increased to 150 ppb (Fig.
3). This suggests a profound loss of stomatal function, although closing remained
faster than opening, even at this high O3.
31
Simulated response curves were generated using mean values of t½
characteristic of the extremes (0 ppb and 125 ppb) of the opening (Fig. 4a) and
closing responses (Fig. 4b). Curves were also generated for the hypothetical case
of t½ = 0, representing instantaneous stomatal response (Figs. 4a,b; long dashed
lines). These curves were integrated to calculate the effect of O3 on cumulative gs
(Fig. 4c) and cumulative water loss (Table 1).
In the case of stomatal opening with t½ = 0, gs increased to maximal values
immediately upon the step change in irradiance (Fig. 4a), allowing cumulative gs
(Fig. 4c; upper dotted line) to increase linearly from the instant of the step change.
This allowed transpiration to begin accumulating immediately at a linear rate,
driven by the product of steady state gs after the step change and VPD. With the
small but finite t½ observed at low O3, accumulation of gs began more slowly and
the rate of accumulation increased over time as stomata opened, becoming linear
with time as stomata neared steady state (Fig. 4c). With the longer t½ at 125 ppb,
the stomatal opening response was even slower. At 10 min after the step change,
cumulative gs at 125 ppb was 54 % of gs at 0 ppb and 40 % of the hypothetical
case. Cumulative gs was strongly affected by stomatal response kinetics (Fig. 4c)
and the hydrologic implications were significant (Table 1).
At low O3, transpiration was reduced by 21 mm month-1 relative to the
hypothetical case. At 125 ppb O3, transpiration was reduced by 46 mm month-1
relative to the hypothetical case, and by 24 mm month-1 relative to low O3 (Table
1).
A similar, but opposing, effect was observed in response to a step reduction
in irradiance leading to stomatal closure. With the hypothetical t½ = 0,
accumulation of gs stopped immediately with the step change (Fig. 4b; long
dashed line). This led to no accumulation of gs following the step change (Fig. 4c).
32
0.4
(a)
Hypothtical response (t1/2 = 0)
0.3
0 ppb
0.2
Stomatal Conductance (mol m-2 s-1)
0.1
125 ppb
0.0
Stomatal Opening
0.4
Stomatal Closing
0.3
0.2
125 ppb
0.1
0 ppb
0.0
Hypotheticalresponse (t1/2 = 0)
(b)
Cumulative Stomatal Conductance (mol m-2)
200
Opening
Hypothetical
t1/2 = 0
(c)
Opening
0 ppb
150
Closing
125 ppb
100
Opening
125 ppb
Closing
0 ppb
50
Closing
Hypothetical
t1/2 = 0
0
0
2
4
6
8
10
Time after step change in PPFD (min)
Figure 4. Simulated time courses of stomatal opening.
Note: (a) and closing (b) in response to a step change in irradiance from 100 to 1600 µmol m-2 s-1
(opening) or 1600 to 100 µmol m-2 s-1 (closing), reflecting experimentally determined mean t½s
constants at representative acute O3 concentrations (0 ppb, solid lines), (125 ppb, short dashed
lines), and under the hypothetical cases of instantaneous stomatal response (t½ = 0, long dashed
lines; (c) effect of acute O3-induced changes in t½s of stomatal opening (dotted lines) and
stomatal closing (solid lines) in response to a step change in irradiance on cumulative stomatal
conductance, for the representative cases shown in panels (a) and(b).
Table 1. Impact of ozone on the half time (t1/2) for stomatal response and resulting water consumption by Pima
Cotton.
Effect of stomatal response kinetics
on Transpiration
Hypothetical
Instantaneous
Response
Ozone
concentration
0 ppb
125 ppb
0 ppb
Relative to t½=0
125 ppb
Relative to
t½=0
125 ppb
Relative to
0 ppb
Stomatal opening
Half-Time (t½)
Cumulative
Transpiration1
0 min
2.06 min
6.42 min
mm hr-1
.44
.32
.18
mm month-1
78
57
32
0 min
2.36 min
4.43 min
mm hr-1
0
.14
mm month-1
0
26
-.14
-.12
-21
-.26
-46
.23
+.14
+.23
+.09
41
+26
+41
+15
-24
Stomatal closing
Half-Time
Cumulative
Transpiration1,2
1
Calculated over the initial 10 minutes following each step change in PPFD, the period of maximum rate of response,
assuming 3 transitions in each direction per hour, LAI = 4 (Grantz et al. 1993), and midday VPD = 0.04 mol
mol-1.
2
Monthly values assume 6 h per day for a 30 d month.
33
34
At low O3, accumulation of gs began at the same rapid rate but the rate of
accumulation decreased over time, becoming linear as stomata closed toward the
new steady state. At 125 ppb, accumulation of gs also began at the same rate,
dictated by steady state gs at the instant of the step change and prevailing VPD.
With the slower closing kinetics, the rate of accumulation declined more slowly
than at low O3. At the end of the 10 min simulation period, the high O3 treatment
had 1.6-fold greater cumulative gs than the no-O3 control, and substantially (in
theory infinitely) more than at t½ = 0.
Slower stomatal closure at low and high O3 increased water use by 26 and
41 mm month-1, respectively, relative to the hypothetical case. High O3 increased
water use by 15 mm month-1 relative to low O3 (Table 1).
The net effect of high O3 exposure on stomatal response kinetics was a
decrease in transpiration by 9 mm month-1, relative to low O3, and by 5 mm
month1, relative to the hypothetical t½ = 0. The presence of a finite t½ at low O3
increased net water use by 5 mm month-1, relative to the hypothetical case.
In sorghum, the response of stomata was not consistent (Fig. 5). The
closing of the stomata was higher than the opening of stomata in response to most
of the O3 concentration, this is difficult to explain biologically. Thus, there may be
other factors causing this in the experimental system.
In cotton, the increase in nocturnal gs suggests specific inhibition of the
closing mechanism by O3, with implications for the kinetics of closure. The t½ for
both opening and closing increased linearly with O3 from 0 ppb to 125 ppb, then
declined sharply at 150 ppb, suggesting a breakdown in stomatal regulation at high
O3. Opening was more sensitive to O3 than closing and was consistently slower
(longer t½), consistent with previous results of Hetherington and Woodward
(2003).
35
Figure 5. Effect of acute O3 exposure in sorghum on the kinetics of stomatal
opening (circles; solid line) and stomatal closing (triangles; dashed line) in
response to a step change in irradiance from 100 to 1600 µmol m-2 s-1 (opening) or
1600 to 100 µmol m-2 s-1 (closing).
Response rate is negatively related to stomatal size (Drake et al., 2013) and
stomatal kinetic parameters are sensitive to plant stress history (Assmann and
Grantz, 1990; Lawson and Blatt, 2014; Pearcy and Way, 2012). Also, an
interaction at the level of signaling has been suggested (Wilkinson and Davies,
2010), in which stress ethylene induced by O3 antagonized responses to abscisic
acid. Abscisic acid is well-known to induce stomatal closure under conditions of
soil or root water deficit. However, ethylene emission in this cultivar of Pima
cotton did not respond to O3 over this range of exposure (Grantz , 2012).
Conclusion
In cotton, the kinetics of both stomatal closure and opening were slowed in
dose dependent manner. Transpiration increased with slower daytime closure but
36
they were more than offset by slower opening. The net effect of O3 was decreased
water use in cotton. The present study confirmed previous observations of sluggish
stomatal responses. The reduction in water loss caused by sluggish stomatal
opening more than compensated for the increase caused by sluggish closure. The
net effect was a decrease in water loss by 9 mm month-1 at 125 ppb. In sorghum,
closing was faster than opening, but there was no effect of ozone on the kinetics.
37
OBJECTIVE II: THE EFFECT AND INTERACTION OF O3
EXPOSURE TO MILD WATER DEFICIT STRESS IN
GROWTH AND PHYSIOLOGICAL PARAMETER
IN PIMA COTTON
Introduction
Soil water deficit (WD) and tropospheric O3 are increasing and projected to
increase in many crop production regions as well as in unmanaged ecosystems
(Field and Van Aalst, 2014). These projections will create new challenges for
production of food, fiber, and bioenergy ( The Royal Society, 2008; Stevenson et
al., 2006; Vingarzan, 2004; Wilkinson et al., 2012). Current and anticipated O3 can
cause direct damage to vegetation (Booker et al., 2009) thus suppressing
agricultural yield (Avnery et al., 2011; Emberson et al., 2009; Lefohn et al., 1988)
and inhibiting growth of native vegetation (Materna, 1984).
Ozone alters plant growth and structure (Heggestad et al., 1988; Heggestad
and Lesser, 1990) and impacts daily maximum gs and photosynthesis (Heath,
1994; Reich and Lassoie, 1984), it also reduces the duration of maximal rates
(Paoletti and Grulke, 2005). Plant WD has impacts similar to those of O3, both
reducing magnitude and duration of daytime gas exchange and suppressing
biomass production (Grantz and Shrestha, 2006; Grantz and Yang, 1996; Tingey
et al., 1971).
Ozone and WD interactions are common but poorly characterized (Fuhrer,
2003, Hoshika et al., 2013; Mansfield, 1973). Antagonistic interactions have been
described, in which effects of O3 are reduced by WD (Temple, 1986; Temple et
al., 1988; Temple, 1990) mediated through stomatal regulation, though synergistic
responses have also been described (Heggestad et al., 1985). Interactions of O3
and WD for biomass productivity and morphological traits are not well
understood.
38
Morphological characters such as harvest index and the spatial and
temporal extent of leaf area display have been heavily selected in modern crop
cultivars (Jin et al., 2010; Long et al., 2006, Morrison et al., 1999; Morrison et al.,
2000) and there may be little scope for further improvement. It has been difficult
to identify optimal stress tolerant phenotypes (Ainsworth et al., 2012;
Hetherington and Woodward, 2003), though maintenance of architecture under
environmental stress may be key to preserving yield under non-optimal conditions
(Betzelberger et al., 2012).
Stomatal conductance is rapidly and easily measured and it is closely
correlated with photosynthetic C assimilation. In C3 plants, such as cotton, gs
effectively regulates exchange of water vapor, O3, and contributes to C
assimilation when radiation is not limiting. Stomatal closing responses are
defenses against both stressors, and are themselves responses to them. Stomatal
closing responses to WD reduce O3 influx (Grantz et al., 1997; Temple, 1986;
Temple et al., 1988; Temple, 1990), while O3 induced closure reduces
transpiration and conserves soil moisture (Hayes et al., 2012). Additionally, there
is biochemical cross-protection against independent stressors (Vannini et al.,
2006), though this is not observed in all cases (Biswas and Jiang, 2011).
It has been considered that increases in gas exchange through modification
of photosynthesis, photosynthetic efficiency, or gs might not translate into
increased ecosystem productivity or crop yield (Evans et al., 1998). Theoretical
arguments (Sinclair et al., 2004) and weak correlations between single leaf
photosynthesis and yield (Kumudini, 2002) support this perspective, as do
observations that photosynthesis in high-yielding cultivars is not consistently
greater than in low-yielding ancestors (Evans and Dunstone, 1970). Nevertheless,
single-leaf gas exchange parameters have in some cases increased with selection
39
(Cornish et al., 1991; Jin et al., 2010; Koester et al., 2014; Morrison et al., 1999).
Enhanced productivity in CO2 enriched environments with elevated
photosynthesis suggests that biochemical enhancement of photosynthesis may also
improve yield (Ainsworth et al., 2002). Enhanced productivity through improved
photosynthetic efficiency may be more effective and more feasible than previously
thought due to recent advances in modeling and molecular techniques (Ainsworth
et al., 2012; Long et al., 2006).
Relationships between the impacts of O3 and WD on gas exchange behavior
at different times of the day are not known. There may be potential for altered
stomatal regulation that avoids periods of high evaporative demand and elevated
O3, contributing to stress tolerance (Dumont et al., 2013). In addition, relationships
between gas exchange behavior at different times of the day and impacts of these
stressors on productivity, biomass allocation, and functional parameters such as
maintenance of plant water relations, have not been characterized.
Therefore, the objective of this study was to investigate the effect and
interaction of O3 exposure to mild water deficit stress in growth and physiological
parameter in Pima cotton.
Materials and Methods
A study was conducted in the research greenhouse at the University of
California, Kearney Agricultural Center (Parlier, CA USA; 103masl; 36.598 ̊N,
119.503 ̊W). The experiment was conducted twice (two run). The first run was
conducted during early summer and the second run was conducted during late
summer of 2013.
40
Plant Material and Growth Conditions
Seeds of cv. S-6 Pima cotton as described in experiment I was also used in
this study and germinated in the same greenhouse using the same methodology as
described earlier. This cultivar of Pima cotton is O3-sensitive (Grantz and Yang,
1996). Seeds were planted on July 10, 2013 for the first run and August 14, 2013
for the second run. At 10 or 12 d after planting (DAP) (for run 1 or 2 respectively),
two pots were transferred to each of 9 cylindrical, Teflon O3 exposure chambers
(Continuously Stirred Tank Reactors; CSTRs; Heck et al., 1978) as previously
described (Grantz et al., 2010; Grantz et al., 2008). Air temperature in the
greenhouse ranged from 15 to 30 C and illumination with natural sunlight was
about 300 mol m−2 s−1 photosynthetic photon flux density (PPFD) at plant level.
Air with appropriate O3 concentrations (12-h means 4, 59, and 114 ppb; was
introduced at one complete air exchange per minute into each CSTR. Ozone was
dispensed from the time of transfer to the CSTRs as daily half-sine waves (7
d/wk), with peak concentration at solar noon (13:00 h). Ozone was generated by
corona discharge (Model SGC-11, Pacific Ozone Technology, Brentwood, CA,
USA) from purified oxygen (Series ATF-15, Model 1242, SeQual Technologies
Inc., San Diego, CA, USA). Air containing the desired O3 concentration was
introduced at one complete air exchange per minute into the orbit of a 120 rpm
circulating fan for uniform distribution. Ozone was regulated in a single CSTR by
a dedicated O3 monitor (Model 49 C, Thermo Fisher Scientific, Waltham, MA,
USA), with computerized feedback control (Grantz et al. 2008). The O3
concentrations in the other CSTRs were maintained proportionally and determined
every 15 min with a separate O3 monitor (Model 49C), through continuously
purged Teflon dust filters and tubing. Ozone monitors were calibrated against a
factory certified calibration unit (Model 306, 2B Technologies; Boulder, CO,
41
USA). Three concentration profiles of O3 were dispensed from the time of transfer
to the CSTRs as daily half-sine waves (7 d/wk). The 12-h mean daylight O3
exposures (0700–1900 h) were 4, 59 and 114 ppb (Grantz et al., 2008).
Field capacity of the pots was determined gravimetrically, after 3 h
drainage. Well-watered plants (WW) were irrigated daily with 600 ml (about field
capacity, determined gravimetrically after 3 h drainage) with automated drip
emitters. Drainage from the pots declined with plant growth. Water-deficit plants
(WD) received an average of 200 ml day-1. In Run 1 this amount was applied
daily, with some drainage initially. In Run 2 pots were returned to field capacity
every third day, with somewhat greater drainage. Plant behavior did not differ
between these water application protocols. After the development of first two true
leaves, pots were fertilized twice a week with a complete fertilizer solution
(Miracle Gro; Scotts Miracle-Gro Products Inc., Port Washington, NY, USA).
Data Collection
Day time (diurnal) gs was measured at one and half hour intervals starting
from 7:30AM to 6 PM on the youngest fully-expanded leaf (YFEL). The night
time (nocturnal) gs was measured at 2AM. In first run, measurement were taken on
two occasions for diurnal gs and on three occasion for nocturnal between 30 and
45 DAP, using a cycling leaf porometer (AP-4; Delta-T Devices, Cambridge, UK).
In the second run, both diurnal and nocturnal gs were taken on four occasion
between 30 to 45 DAP, using the cycling leaf porometer. The same leaf was used
for all measurements on each day, avoiding major veins, and the next youngest
leaf as it became fully expanded was used on subsequent days. An index of
stomatal responsivity to O3 was calculated from mean gs in each O3 and water
42
treatment (WW or WD), as the stomatal response from low O3 to high O3
normalized by gs at low O3.
Chlorophyll content of the plants was measured with a young fullyexpanded leaf with a portable chlorophyll meter (Minolta SPAD-502, Spectrum
Technologies, Plainfield, IL, USA) in 3 occasion for each run in between 30 to 45
DAP.
Plants were destructively harvested at 45 and 47 DAP for the first and
second run, respectively. Plants were separated into leaves (including the petioles),
stems, and roots. Leaf area was determined on pooled leaves from individual
plants, using a belt driven leaf area meter (LI 3100; LiCor Inc. Lincoln, NE,
USA). Roots were washed thoroughly in cool water to remove potting medium.
Dry weights of plant components were determined using an electronic balance
(Model 200, Mettler-Toledo, Columbus, OH, USA; capacity 200 g, precision 1 ×
10-4 g) after drying with forced air at 60°C to constant weight for a week.
Allometric parameters were calculated on an individual plant basis.
Experimental Design and Data
Analysis
The experiment was replicated in three blocks of CSTRs aligned parallel to
the windows and cooling fans in the greenhouse to isolate location effects, and
performed twice. One CSTR in each block was exposed to each of the three O3
concentrations. The experimental design was a split-plot arranged as randomized
complete block. The main treatment was O3 concentration and the sub-treatment
was water application rate. The individual CSTRs were considered as a replicate.
Data were analyzed using the general linear model procedures (PROC
GLM) of SAS (SAS v. 9.2.1). Assumptions of ANOVA were tested and data that
failed to meet the normality assumptions of ANOVA were log-transformed prior
43
to analysis. This was only necessary for the gs data. Ozone treatments and
irrigation sub-treatments were considered fixed factors and block and runs were
considered as random factors. Interactions between the various factors were also
tested.
When the effect of a treatment was significant by F test (p < 0.05), mean
separation was determined using Fisher’s Least Significant Difference (LSD) test.
Runs yielded consistent results and had no interactions with any of the factors.
Therefore, data for the two runs were combined. In the case of gs, the interaction
between water regime and O3 was significant, so effects of water was further
analyzed within each level of O3. For all other parameters the water by O3
interaction was not significant, so that experiment-wise effects of O3 were
evaluated across both water application treatments. All data are presented as
means ± standard errors (s.e.), calculated from non-transformed data.
Results and Discussion
Stomatal Conductance
Pima cotton exhibited a bell-shaped diel time course of gs (Fig. 6a, circles).
Under control condition of low O3, the well watered (WW) plants were relatively
stable with maximum gs occurring around midday. The magnitude of gs was
reduced throughout the day by both high O3 (Fig. 6a, triangles) and by WD (WD;
cf. Fig. 6a,b; circles). Under all conditions, values of gs converged with stomatal
closure at the end of the day. Midday gs were considerably greater than that in the
morning, early evening, or at night (Fig. 6). The sensitivity of gs to O3 exposure
varied with time of day, with relative closure increasing from morning through
late afternoon (Fig. 6) and decreasing toward the evening. As both magnitudes and
44
(a)
600
WW
LO3
200
HO3
-2
-1
Conductance (mmol m s )
400
0
(b)
WD
(
600
LO3
400
200
HO3
0
0
2
4
6
8
10
12
14
16
18
20
22
24
Time of day
Figure 6. The effect of chronic ozone (O3) exposure on the diel timecourse of
stomatal conductance (gs) in Pima cotton (a) under well-watered (WW) or b waterdeficit (WD) conditions. Plants were exposed to the low O3 treatment (LO3:4 ppb;
12 h mean) or the high O3 treatment (HO3;114ppb; 12 hr mean) mean ±S.E.
45
relative responses to O3 and WD were greater at midday, responses at this time
dominated the daily integrated response of gas exchange.
The WD treatment alone was less effective than high O3 alone in reducing
midday gs. High O3 suppressed gs by about 65% in WW and 35% in WD.
However, in the presence of high O3, the addition of WD did not further reduce gs,
yielding similar time courses for the high O3 treatment under both WW and WD
conditions.
Midday gs and transpiration were reduced by 75% under WW conditions
and by 60% under WD conditions (Fig. 6; Table 2). This was projected to reduce
water loss by 388 mm month-1 and 165 mm month-1, respectively (Table 2).
At night, gs increased with high O3 exposure, (Fig. 6a, b; 02:00) under both
WW and WD conditions. At high O3, nocturnal gs and transpiration increased
83% under WW conditions and 151% under WD conditions (Fig. 6; Table 2). This
was projected to lead to increases in stand water loss of 8.2 mm month-1 and 10.4
mm month-1, respectively (Table 2). The increase in nocturnal water use was
hydrologically significant, but considerably smaller than the decrease in daytime
water use. The net effect of O3 –induced changes in the diel course of gs was a
substantial reduction in water loss of 380 mm month-1 under WW conditions and
of 155 mm month-1 under WD conditions (Table 2).
Chronic O3 exposure and the irrigation regime reduced daytime gs. Water
deficit had less effect at the high O3 concentration. The impact of O3 was smaller
in WD plants. Similar results were observed in upland cotton (G. hirsutum)
(Temple, 1986; Temple, 1990). Reduced daytime gs is protective against O3 and
WD by reducing uptake when peak O3 occurs and water loss when peak VPD
occurs (Cavender et al., 2007; Massman et al., 2000; Paoletti and Grulke, 2005).
Both factors truncated the duration of maximal daytime gas exchange. These O3
Table 2. Impact of ozone and irrigation regimes on daytime and nocturnal steady state stomatal conductance and
resulting water consumption by Pima Cotton.
Time of Day (hrs)
02:00
Ozone Concentration
(ppb)
Irrigation
regime2
4
WW
114
4
114
13:30
WD
4
WW
114
4
114
WD
Stomatal
Conductance
Transpiration1
Effect on Transpiration2
mmol m-2 s-1
mm hr-1
mm month-1
11
0.055
20
0.101
8
0.038
19
0.096
560
5.75
140
1.44
298
3.06
120
1.23
+8.2
+10.4
-388
-165
1
Source: Assuming LAI = 4 (Grantz et al. 1993); VPD = 0.02 mol mol-1 at night and 0.04 mol mol-1 at midday.
2
WW = well watered (600 ml pot-1 day-1); WD = water deficit (200 ml pot-1 day-1).
3
Difference between High O3 and Low O3, assuming 3 h of average midday transpiration and 6 h of nocturnal
transpiration per day, for a 30 day month
46
47
effects are associated with reduced net photosynthesis and increased intercellular
CO2 (Aphalo and Jarvis, 1993; Paoletti and Grulke, 2005), reduced allocation to
roots and associated plant hydraulic conductance (Grantz et al., 2006; Heggestad
et al., 1985; Nikolova et al., 2010), and direct impacts on guard cell metabolism
(Torsethaugen et al., 1999).
Nocturnal gs was increased substantially at high O3 relative to low O3, by
83% in WW and 151% in WD. This is consistent with large relative responses
observed in other species (Grulke et al., 2004; Grulke et al., 2007; Hoshika et al.,
2013; Matyssek et al., 1995; Wieser and Havranek, 1993, 1995). In blue oak and
black oak, chronic O3 exposure increased nocturnal gs to about 16% and 30% of
daytime maxima (Grulke et al., 2007a). In birch, nocturnal gs contributed about
10% of total transpiration at low O3 (Matyssek et al., 1995), increasing to 15% in
high O3. Pima cotton has been selected for higher gs than is observed in most forest
species (Lu and Zeiger, 1994). This could potentially alter the relative impact of
daytime and nocturnal stomatal responses.
In Pima cotton, the reduction in water loss in the daytime more than
compensated for the increase at night, reducing transpiration under both WW and
WD conditions, by 380 and 155 mm month-1, respectively.
Leaf Parameters
Leaf chlorophyll content, measured in SPAD units, declined substantially at
high O3 (Table 3). In contrast to gs, leaf pigmentation was greater in the WD
leaves than in the WW leaves at all levels of O3. Convergence of SPAD at high O3
did not lead to a significant O3 and WD interaction. Some bronze discoloration on
lower leaves, an indication of O3-induced early senescence, was observed in high
O3, but neither necrosis nor accelerated abscission was observed.
48
Under WW conditions, leaf mass per area (LMA) declined substantially at
moderate O3 and only marginally further at high O3, while WD leaves did not
respond to O3 at any level (Table 3). Initial values of LMA at low O3 were greater
in WW than WD, but the sensitivity to O3 in WW reduced LMA to levels
comparable to WD at moderate O3, and to levels below WD at high O3. Large
variability was observed in this derived parameter. There was no interaction
between the water level and O3 (Table 3). Mean separation between the O3
treatments was performed with experiment-wise F test at p = 0.0797 (Table 3); at
this significance level, there was difference in LMA between the high and low O3
treatments. Leaf biomass and leaf area per plant (Table 3) exhibited responses
similar to those of shoot biomass (Table 4), declining substantially at high O3. The
effect of WD was not pronounced.
No necrotic lesions on the leaves were observed in this experiment, but
some bronze stippling and accelerated senescence were observed on lower leaves
under high O3 concentration. Although necrotic lesions are commonly observed
under high O3 concentration (Goumenaki et al., 2010; Pell et al., 1997; Sarkar et
al., 2010), they were not observed in this study which could be because of the use
of juvenile plants and the short O3 exposure duration compared to the other
studies. Ozone-induced abscission was also not observed in this study. Responses
of SPAD to O3 concentration were closely related to responses of gs at midday but
less so to responses of gs in early morning or late afternoon. SPAD was not driven
by leaf anatomical changes, as LMA was not closely related to midday gs and was
less responsive than SPAD to O3, particularly in the WD treatment.
Leaf pigmentation was greater in WD than in WW leaves at all levels of O3,
in contrast to gs. This pattern reflected the relatively mild WD treatment, as more
severe WD decreases leaf chlorophyll (Parida et al., 2007). SPAD may represent a
Table 3. Impact of ozone and irrigation regime on leaf pigmentation and leaf properties of Pima cotton.
Chlorophyll conc.3
Total leaves3
Leaf area3
Leaf dry wt. 3
Leaf mass area3
SPAD units1
(leaves plant-1)
(cm² plant-1)
(g plant-1)
(g m-2)
4
34.7a
10a
712.2a
2.59a
38.4a
59
34.7a
10a
699.5a
2.47a
35.0ab
114
29.3b
6b
216.7b
0.72b
34.0b
WW
33.8A
9A
556.1A
1.97A
35.7A
WD
32.0B
8A
529.5A
1.89A
36.0A
Ozone
0.0387
0.0067
0.0006
0.0043
0.0797
Irrigation
0.0371
0.6725
0.7111
0.691
0.8870
Ozone X Irrigation
0.8559
0.5186
0.8269
0.5892
0.2701
Treatment
Ozone conc. (ppb)
Irrigation regime2
P-values
1
Mean of 6 d
WW = well-watered (600 ml pot-1 day-1); WD = water deficit (300 ml pot-1 day-1).
3
Means within a column for ozone concentration followed by the same lowercase letters and the same uppercase
letters for the irrigation regime are not significantly different at a 0.05 level of significance according to Fisher’s
Least Significant Difference (LSD) test.
2
49
50
useful surrogate for O3-induced changes in midday gs but not for WD-induced
changes, and not for stomatal responses in morning or afternoon.
Shoot Parameters
Total plant biomass was dominated by the shoot (Tables 4, 5). Moderate O3
caused little response but a substantial decline in shoot biomass was observed at
high O3. The irrigation regime had no effect on shoot biomass. Shoot biomass was
approximately evenly divided among stem and leaf components. The responses of
number of leaves per plant (Table 3) and plant height (Table 4) to O3 differed from
responses of biomass and leaf area.
The effect of O3 on both was significant (Tables 3, 4), driven by the
response to high O3 relative to moderate O3, as above. In both WW and WD
treatments, plant height increased from low to moderate O3 but declined at high
O3.
The impact of these responses on plant performance is suggested by leaf
insertion rate (Table 3), the inverse of internode length. The vertical density of
leaves in the canopy is a component of leaf area index (LAI) and radiation
interception. The leaf insertion rate declined with increasing O3 concentrations in
both WW and WD treatments. However, the irrigation regime had no effect on the
leaf insertion rate. The response of leaf insertion rate was not driven by extension
growth but rather by leaf initiation.
Root Parameters
Production of root tissue was inhibited by an increase in O3 concentrations
(Table 5) in a pattern similar to that of shoot biomass (Table 4). However, similar
to shoot biomass, the irrigation regime had no effect on root biomass. Two
allometric parameters were derived to further assess the impacts of O3 and WD on
Table 4. Impact of ozone and irrigation regime on shoot properties of Pima cotton.
Plant height2
Stem wt.2
Leaf insertion rate2
Shoot wt. 2
(cm)
(g plant-1)
( leaves cm-1)
(g plant-2)
4
42.2ab
2.30a
0.24a
4.9a
59
46.0a
2.42a
0.22a
4.9a
114
37.3b
0.54b
0.17b
1.3b
WW
42.2A
1.81A
0.21A
3.7A
WD
41.5A
1.70A
0.20A
3.59A
Ozone
0.0123
0.0359
0.0317
0.0148
Irrigation
0.7558
0.6254
0.6867
0.6535
Treatment
Ozone conc. (ppb)
Irrigation regime1
P-values
Ozone X Irrigation
0.3676
0.6994
0.8668
0.6407
1
-1
-1
-1
-1
WW = well-watered (600 ml pot day ); WD = water deficit (300 ml pot day ).
2
Means within a column for ozone concentration followed by the same lowercase letters and the same uppercase
letters for the irrigation regime are not significantly different at a 0.05 level of significance according to Fisher’s
Least Significant Difference (LSD) test.
51
Table 5. Impact of ozone and irrigation regime on root properties of Pima cotton.
Root wt.2
Total biomass2
(g plant-1)
(g plant-1)
4
0.90a
5.8a
0.19a
13.8a
59
0.80a
5.7a
0.16b
11.6a
114
0.17b
1.4b
0.14b
8.3b
WW
0.64A
4.43A
0.16A
11.6A
WD
0.61A
4.2A
0.15A
10.8A
Ozone
0.0182
<0.001
0.0015
0.0002
Irrigation
0.6686
0.6576
0.3363
0.4173
Treatment
Root Shoot Ratio2
Root mass per leaf area2
(g plant-2)
Ozone conc. (ppb)
Irrigation regime2
P-values
Ozone X Irrigation
0.3795
0.6213
0.4605
0.5303
1
-1
-1
-1
-1
WW = well-watered (600 ml pot day ); WD = water deficit (300 ml pot day ).
2
Means within a column for ozone concentration followed by the same lowercase letters and the same uppercase letters
for the irrigation regime are not significantly different at a 0.05 level of significance according to Fisher’s Least
Significant Difference (LSD) test
52
53
the plant parameters, i.e. root to shoot biomass ratio and root biomass per leaf
area. The root to shoot biomass ratio (Table 5) was similar between the plants
under the two irrigation regimes and no interaction occurred between the O3
concentration and the irrigation regime. Root mass per leaf area is a similar but
more functionally relevant allometric parameter. This parameter reflects the
balance between water acquisition and transpirational water loss. This derived
parameter was also proportionally reduced by increase in O3 concentrations.
However, similar to the other parameters, the irrigation regime had no effect on
root mass per leaf area and there was no interaction between O3 concentration and
the irrigation regime either.
The responses of biomass and leaf area were closely linked, and both were
similar to responses of gs observed at midday. The dominant portion of total plant
biomass was located in the shoot. Ozone exposure generally reduces shoot
biomass (Heggestad et al., 1988; McLaughlin et al., 1982; Miller, 1988; Pell et al.,
1994), and the present observations are consistent with previous evidence in
upland and Pima cottons (Grantz and Yang, 1996; Miller, 1988; Oshima et al.,
1979; Temple et al., 1988; Temple, 1990). Responses of root biomass to O3 were
also similar to responses of shoot components and midday gs consistent with other
studies (Cooley and Manning, 1987; Grantz et al., 2006; Grantz and Yang, 1996;
Kostka et al., 1993). Reduced allocation to roots has been associated in this
cultivar with reduced translocation of carbohydrate from source leaves (Grantz
and Farrar, 1999; Grantz and Farrar, 2000). However, the irrigation regime had no
effect on the shoot and root biomass. This could probably be due to the following
reasons. The WD treatment was not severe enough to cause the anticipated level
of stress, the restriction of the root system to a small volume of soil in a small pot
size did not cause a big enough difference between the two irrigation regimes to
54
allow differences in plant biomass, the duration of the experiment was not long
enough to cause pronounced effects on the biomass of the plants, and/or the more
pronounced effect of O3 on the plants masked the effect of irrigation regimes.
Again, the results of the irrigation regimes may have been different under
prolonged exposure conditions. These data were not inconsistent with Hypothesis
3, suggesting that midday measurements of stomatal responses reflect broader
impacts of O3 and WD on plant development.
Plant height did not respond similarly to biomass and leaf area. It was
reduced by WD at low and moderate O3 but protected by WD at high O3, and was
increased in moderate O3 in both WW and WD, relative to both low and high O3.
This reflects the integration of etiolation and reduced productivity observed at
moderate to high O3 as observed in other studies (Kostka et al., 1993; Leone and
Brennan, 1975; Tingey, Heck, et al., 1971). Leaf insertion along the stem, the
inverse of internode length, declined with O3 exposure, driven by leaf initiation
rather than by stem elongation. Maintenance of leaf initiation may contribute to
yield stability in stress-prone environments.
Reduced root growth reduces soil exploration and impairs root hydraulic
conductance (Grantz and Yang, 1996; Heggestad et al., 1985; Lee et al., 1990;
McLaughlin et al., 1982). This might increase sensitivity to soil water deficit
(Heggestad et al., 1985) and enhance xylem cavitation (Tyree and Sperry, 1988),
as observed in soybean (Heggestad et al., 1985). The functional balance between
water acquisition and water loss to transpiration, reflected in the root to shoot
biomass ratio and root mass per leaf area, was significantly reduced by exposure to
O3 under both WW and WD conditions. This has been described previously in
Pima cotton (Grantz and Yang, 1996) and in maple and oak (Ren and Sucoff,
1995; Yang and Tyree, 1993, 1994), beans (Fiscus and Markhart, 1979) and
55
sugarcane (Meinzer and Grantz, 1990). In many species, including cotton (Grantz
and Yang, 1996, Temple, 1986, Temple, 1990), alfalfa (Temple et al., 1988), and
red spruce (Lee et al., 1990), strong positive relationships between stomatal and
hydraulic conductance stabilize plant water potential as both change with
development, water deficit, or O3 (Grantz and Farrar, 1999, Lee et al., 1990,
Meinzer and Grantz, 1990, Meinzer, 2002). Although root growth is generally
reduced by O3 (Grantz et al., 2006) and protected or even increased by WD (Sharp
and Davies, 1989), there was no protective antagonism observed at moderate or
high O3 in the present study. These functional parameters were not closely related
to gs observed at midday or other period.
Conclusion
The present study confirmed previous observations of sluggish stomatal
responses and incomplete nocturnal closure following plant exposure to high
concentrations of O3. While this was predicted to increase regional transpiration
and reduce stream flow in mixed deciduous forests (McLaughlin et al., 2007; Sun
et al., 2012; Uddling et al., 2009), in Pima cotton the net effect, considering both
daytime and nocturnal responses, and both opening and closing responses, was a
reduction of simulated transpiration. The reduction in water loss caused by
sluggish stomatal opening more than compensated for the increase caused by
sluggish closure. The net effect was to decrease water loss by 9 mm month-1 at 125
ppb. In SOYFACE a consistent decrease in canopy water loss was observed with
increasing O3 under field conditions (VanLoocke et al., 2012).
These conclusions are conservative in that O3-induced reductions in leaf
area and transpiration following chronic O3 exposure, are not considered. In
temperate and boreal forests, rising CO2 decreased transpiration by a stomatal
56
mechanism and increased runoff (Gedney et al., 2006; Keenan et al., 2013),
though these stomatal effects may be offset by longer-term changes in leaf area
(Torngern et al., 2015). In forest systems, increased in leaf area may compensate
for reduced gs caused by chronic CO2 exposure (Torngern et al., 2015), despite
some evidence of decreased stand transpiration. No greenhouse and laboratory
study can be directly applied in field conditions. Stomata respond to multiple
stimuli in the field, so that the fixed frequency of step changes in PPFD imposed
in the simulation is a necessarily simplified scenario. Energy balance and leaf
boundary layer conductance will differ under field conditions. However, an
increase in leaf temperature of 3ºC due to decreased gs, a similar magnitude to that
observed in SOYFACE (VanLoocke et al., 2012) over a similar range of O3. This
would increase midday transpiration by less than 20%, and incorporation of
realistic single leaf boundary layer conductance in the field (Grantz and Vaughn,
1999) would reduce midday water loss by 10 - 16%.
This study suggested that species and environmental differences will
complicate prediction of O3 and WD impacts on regional hydrology, requiring
further study in a broad range of systems.
57
OBJECTIVE III: THE EFFECT OF O3 EXPOSURE AND MILD WATER
DEFICIT STRESS IN GROWTH AND PHYSIOLOGICAL RESPONSES
IN SELECTED AMARANTHUS SPECIES (PALMER AMARANTH
AND COMMON WATERHEMP)
Introduction
Global climate change caused by increased emission of greenhouse gases is
altering hydrological systems. Tropospheric O₃ is a major air pollutant and an
important anthropogenic stressor in agricultural cropping systems. Similarly, water
availability for irrigation of agricultural crops continues to be a challenging issue.
Crop production is challenged by both air pollution and weed pressure in many
irrigated agricultural regions (Grantz et al., 2010b). The effect of O3 and weeds on
crops and their interaction needs to be characterized for effective weed
management strategies to sustain or improve crop yield (Shrestha and Grantz,
2005). Studies have reported that O₃ can have differential effects on crops and
weeds and thereby alter crop-weed competition dynamics. Some weeds such as
black nightshade (Solanum nigrum), horseweed (Conyza canadensis) (Grantz et
al., 2008), and yellow nutsedge (Cyperus esculentus) (Shrestha and Grantz, 2005)
have been found to be tolerant to O₃ and in some cases more competitive with
crops under elevated O₃ conditions. Ozone impacts on nocturnal gs have been
suggested to increase transpiration and alter hydrology in some species such as in
Pinus Ponderosa (Grulke et al., 2004) and Betula pendulata (Matyssek et al.,
1995). However, it is not known if similar responses occur in common agricultural
weed species. Furthermore, the combined effect of O₃ and moisture deficiency on
agricultural weeds and its effect on landscape hydrology are not known.
As stated earlier, availability of water for irrigation continues to be a
challenging issue in the Central Valley of California (Quinn et al., 2013). In recent
years, this challenge has been further aggravated by one of the most severe
58
droughts on record in this area (State of California, 2015). To address the issue of
reduced water availability for irrigation, research is being conducted on regulated
deficit irrigation (RDI) in several crops in California (Costello and Patterson.,
2012; Stewart et al., 2011). Regulated deficit irrigation is the application of water
below evapotranspiration (ET) requirements (Kriedemann and Goodwin., 2003)
and this practice can be helpful in coping with situations of limited water supply
(Fereres and Soriano., 2007). However, there are no studies that have assessed the
combined effect of O3 and RDI on crops and weeds. Such a study is needed in the
current context of the Central Valley.
Amaranthus species commonly referred to as “pigweeds” are among the
most troublesome weeds in many crop production systems (Jha et al., 2008).
Among the pigweeds, Palmer amaranth (Amarahtus palmeri S. Wats.) is
considered as one of the most problematic one in annual cropping systems causing
great economic damage in agronomic crops throughout the U. S. and Canada as
well as in other areas of the world. This species is now invading perennial
cropping systems such as orchards and vineyards in the San Joaquin Valley (SJV)
of California. It is a broadleaf, dioecious species (Grichar, 1997) with small seeds
and is a short-lived C4 summer annual species. It is difficult to manage in
agronomic crops because of extended germination times, relatively fast growth,
high fecundity, and long seed viability. It is an erect, branched plant, and can reach
up to 3 to 4 m in height (Horak and Peterson, 1995). It is highly competitive with
crops for light, nutrient, water, and space due to its high water use efficiency
(Morgan et al., 2001) and allelopathic potential (Morgan et al., 2001). Male and
female plants can be distinguished by the prickly inflorescence as the mature
female inflorescences are prickly to the touch due to the presence of stiff bracts.
59
As Palmer amaranth is rapidly expanding and is very competitive with
crops and is also escaping control by several herbicides (Heap, 2015), a better
understanding of its biology and ecology is important to understand to develop
management strategies (Nordby and Hartzler, 2004). Female Palmer amaranth
plants have been reported to produce 200,000 to 600,000 seeds which allows for a
rapid spread of this species (Massinga et al., 2001). Palmer amaranth which has
high light-saturated photosynthetic rates is well adapted to elevated
photosynthetically active radiation (PAR) environments which enables the plants
to maintain competitive growth rates (Jha et al., 2008). This species also has the
ability to lower their light-saturated photosynthetic rates and light compensation
points by increasing leaf chlorophyll content when shaded (Jha et al., 2008). The
severity of the problem with Palmer Amaranth has further increased with the
evolution of herbicide-resistant biotypes (Horak and Peterson, 1995; Jha et al.,
2008).
Palmer amaranth has evolved resistance to four herbicide modes of actions.
Populations resistant to pendimethalin were noted in South Carolina in 1989
(Gossett et al., 1992). Pendimethalin is widely used as a preemergence herbicide
for weed control in Georgia in crops such as cotton, peanut (Arachis hypogaea L.),
and soybean (Glycine max L. Merr.). Resistant populations of Palmer amaranth to
Acetolactate synthase (ALS) inhibitor herbicides was discovered in 1995 in
Kansas (Horak and Peterson, 1995), Similarly, triazine-resistant Palmer amaranth
populations was initially reported in Texas in 1993 and in Kansas in 1995
(Peterson, 1999). The fourth mode of action to which Palmer amaranth has
evolved resistance to is glyphosate. Glyphosate provides broad-spectrum weed
control and is also used in genetically modified glyphosate–tolerant crops.
Glyphosate-resistant (GR) populations of Palmer amaranth was first reported in
60
Georgia in 2006 (Culpepper et al., 2006) and since then has been confirmed in 27
states in the U.S. (Heap, 2015). Horak and Loughin (2000) evaluated four different
Amaranthus species and reported that Palmer amaranth produced more dry
biomass than common waterhemp (Amaranthus rudis J. D, Sauer), redroot
pigweed (A. retroflexus L.), and tumble pigweed (A. albus L.). Additionally, the
leaf area and growth rate (in terms of increase in height per growing degree day of
young Palmer amaranth plants was at least 50% greater than that for the other
Amaranthus species early in the growing season. This gives Palmer amaranth
considerable competitive advantage and gives growers a smaller window of time
for optimal control compared to other broadleaved weeds. Therefore, the effective
control of this species needs to begin with an understanding of its biological and
ecological characteristics.
Common waterhemp is another major weed in field crops and is becoming
a major threat in field crops throughout the Midwestern U.S. (Heap 2015). This
species is most problematic in Midwest soybean and corn (Zea mays L.)
production systems (Legleitr and Bradley, 2008). Common waterhemp has been
listed as the most encountered and troublesome weed in soybean in Missouri as
well as the most encountered broadleaf weed in corn and soybean in Illinois
(Hager et al., 2009).Especially, GR populations of common waterhemp has
become a serious threat to the sustainability of no-till farming practice in the
Midwestern U.S (Cordes et al., 2004; Smith and Hallett, 2006). Common
waterhemp is dioecious, and, therefore, plants are forced to outcross. Furthermore,
female plants are prolific seed producers. It is a prolific seed producing species,
able to produce about 1.5 times more seed than most other species in the
Amaranthus genus (Sellers et al., 2003). On average a common waterhemp plant
generally produces about 250,000 seed, although some plants can produce as
61
many as 1,000,000 seeds when growing under optimum conditions in
noncompetitive environments (Legleiter and Bradley, 2008; Sellers et al., 2003).
These factors increase the potential for genetic variability among common
waterhemp populations and lead to difficulty in its control by herbicides (Patzoldt,
2005). Common waterhemp has been reported to be resistant to more than three
unique sites of herbicide action in the U.S. including ALS inhibitors (Foes et al.,
1998), PPO inhibitors, PSII inhibitors, HPPD-inhibitors, and glycine (Patzoldt et
al., 2002). Glyphosate-resistant populations of common waterhemp have been
reported from several states of the U.S. (Heap, 2015). Glyphosate-resistant
populations were first identified in the U.S. in Missouri in 2008 (Legleiter and
Bradley, 2008) and GR populations of this weed has now been reported from
thirteen states.
Common waterhemp is a problematic weed in Midwestern U.S cropping
systems. Although common waterhemp is not a widespread weed in California, it
has been reported in some counties such as San Diego, Santa Barbara, and
Sacramento (USDA NRCS, 2014). Poor control of common waterhemp was
reported in roadsides in San Joaquin County, CA in 2008 (R. Milller, personal
communications). A preliminary study on seeds collected from this location
showed that the plants were susceptible to glyphosate. However, some bigger
plants (>15 cm) survived the label rate of glyphosate (A. Shrestha, personal
communication). Due to its ability to spread quickly, common waterhemp could
potentially be a problematic weed in the future in Central valley.
Furthermore, both Palmer amaranth and common waterhemp are C4 species
that use water efficiently (Black et al., 1969). It has been reported that Palmer
amaranth tolerated moisture stress better than corn, a C4 crop (Aldrich and
Kremer, 1997). Therefore, studies addressing the effect of O3 and water stress
62
would likely provide important information for better description of the ecological
basis of some problematic Amaranthus weed species such as Palmer amaranth and
common waterhemp, and help in understanding their invasiveness and agronomic
success in an area prone to high O3 conditions in the summer and arid conditions
such as the Central Valley of California. Therefore, the objective of this study was
assess the combined effect of O₃ and two different moisture regimes in two
selected Amaranths species common waterhemp and Palmer amaranth.
Materials and Methods
The study was conducted in the same research greenhouse described earlier
using similar protocols as in experiment II. Studies on common waterhemp and
Palmer amaranth was conducted in summer 2013 and 2014, respectively. Some of
the experimental protocols used in the two species was not similar; therefore, the
methodology is described separately for each species.
Plant Material and Growth Conditions
Common waterhemp. Seeds of common waterhemp were collected from a
roadside in Stockton, CA in 2008, whereas seeds of the same cotton cultivar
described earlier were used for this study. The experiment was conducted twice in
the summer of 2013. Seeds of common waterhemp were planted in moist
commercial seedling mix in plastic germination trays on June 29 and August 4,
2013 in the first and second run, respectively. Once the seedlings emerged and
produced a true leaf, they were transplanted into plastic pots (870 ml; 110mm
square x 125 mm deep; Dillen Products, Middlefield OH) containing moist
commercial potting mix (Earthgro Potting Soil, Scotts Company, Marysville, OH).
The transplanting was done on July 10 and August 13, 2013 in the first and second
63
run, respectively. When the plants developed 5 to 7 leaves, they were transferred
into controlled O3 environment CSTRs as previously described by Grantz et al.,
(2008) on July 10 and August 26, 2013 in the first and second run, respectively.
Palmer amaranth. Seeds of Palmer amaranth were collected from a corn
field in Fresno, CA (36°29.326’N and 119°57.590W) in 2013. Seeds were
scarified to break the hard seed coat to enable germination. Two-hundred seeds
were placed into 50 ml plastic centrifuge tube with cut pieces of double-sided
sandpaper and were shaken for 20 s. The seeds were then planted in plastic trays
containing a (2:1) mixture of a commercial growing media (sphagnum peat moss,
coarse grade perlite, gypsum, dolomitic lime) and palm and cactus mix (sand and
perlite) on 4 April and 6 June 2014, for the first and second run, respectively.
After the seedlings emerged, they were thinned to one plant per pot. The plants
were grown initially on an open bench in the research greenhouse. Once the plants
reached the 5- to 6-leaf stage, they were transferred to the controlled O3 CSTRs on
May 15 and June 21, 2014 in the first and second run, respectively.
Both experiments were conducted in the same CSTRs described in
Experiment II. Ozone concentration followed a half-sine wave during daylight
hours, 7 d wk-1. Voltage to the O3 generator was regulated by feedback from the
exit stream of a master CSTR (Model 41C; Thermo Electron Corp.; Franklin MA,
USA), calibrated against an O3 calibration unit (Model 306; 2B Technologies,
Boulder, CO, USA). The remaining CSTRs were controlled proportionally and
monitored with a separate analyzer (Model 41C) (Grantz et al., 2010). Three
concentration profiles of O3 were dispensed from the time of transfer to the
CSTRs as daily half-sine waves. The 12-h mean daylight O3 exposures (0700–
1900 h) were 4, 59 and 114 ppb (Grantz et al., 2008).
64
Irrigation and Fertilization
Common waterhemp. Two plants of common waterhemp (one for each of
the irrigation treatments) were grown in each chamber in a total of nine chambers.
Field capacity of the pots was determined gravimetrically, after 3 h of drainage.
Well-watered plants (WW) were irrigated daily with 600 ml (about field capacity,
determined gravimetrically after 3 h drainage) with automated drip emitters.
Drainage from the pots declined with plant growth. Water-deficit plants (WD)
received an average of 200 ml day-1. In Run 1 this amount was applied daily, with
some drainage initially. In Run 2 pots were returned to field capacity every third
day, with somewhat greater drainage. Plant behavior did not differ between these
water application protocols. Plants were fertilized only once with a complete
fertilizer complete fertilizer solution (Miracle Gro; Scotts Miracle-Gro Products
Inc., Port Washington, NY, USA) on August 1 and August 30, 2013 in the first
and second run, respectively. The first run was conducted in early summer and the
second run was conducted in late summer of 2013.
Palmer amaranth. Two plants of Palmer amaranth (one for WW and one for
WD) were grown in each chamber in a total of nine chambers. Field capacity of
the pot was determined gravimetrically, after 3 h of drainage. The volumetric
water content of the field capacity was measured with an EC-5 moisture sensor
(Decagon devices) connected with data logger in every half an hour. When the
volumetric water content in the soil reached to 75% of field capacity, WW plants
were irrigated manually to saturated levels. Similarly, the WD plants were
irrigated when the volumetric water content in the soil reached below 25% of field
capacity throughout the study after placement in the CSTRs. The volumetric water
content was monitored every day. Plants were fertilized only once with a complete
65
fertilizer solution (Miracle Gro; Scotts Miracle-Gro Products Inc., Port
Washington, NY, USA) on May 24 and July 5, 2014 in the first and second run,
respectively. The first run was conducted in early summer and the second run was
conducted in late summer of 2014.
Data Collection
Common waterhemp. Day time (diurnal) stomatal conductance (gs) was
measured at one and half hour intervals starting from 7:30 AM to 6 PM on the
youngest fully-expanded leaf (YFEL). The night time (nocturnal) gs was measured
at 2 AM. In the first run, measurement were taken on four occasions for diurnal gs
and on three occasion for nocturnal gs between 40 and 55 d after planting (DAP),
using a cycling leaf porometer (AP-4; Delta-T Devices, Cambridge, UK). In the
second run, both diurnal and nocturnal gs were taken on four occasion between 40
to 55 DAP. The same leaf was used for all measurements on each day, avoiding
major veins, and the next youngest leaf as it became fully expanded was used on
subsequent days. An index of stomatal responsiveness to O3 was calculated from
mean gs in each O3 and water treatment (WW or WD), as the stomatal response
from low O3 to high O3 normalized by gs at low O3.
Chlorophyll content of the plants was measured on a young fully-expanded
leaf with a portable chlorophyll meter (Minolta SPAD-502, Spectrum
Technologies, Plainfield, IL, USA) on 3 occasion for each run between 40 to 55
DAP.
Plants were destructively harvested at 56 and 55 DAP in the first and
second run, respectively. Plants were separated into leaves (including the petioles),
stems, and roots. Leaf area was determined on pooled leaves from individual
plants, using a belt driven leaf area meter (LI 3100; LiCor Inc. Lincoln, NE,
66
USA). Roots were washed thoroughly in cool water to remove potting medium.
Dry weights of plant components were determined using an electronic balance
(Model 200, Mettler-Toledo, Columbus, OH, USA; capacity 200 g, precision 1 ×
10-4 g) after drying in a forced- air oven at 60°C to constant weight for a week.
Allometric parameters were calculated on an individual plant basis.
Palmer amaranth. Day time (diurnal) gs was measured at one and half hour
intervals starting from 7:30 AM to 6 PM on the youngest fully-expanded leaf
(YFEL). The night time (nocturnal) gs was measured starting from 7:30 PM to
6:00 AM on the YFEL with 24 h difference between daytime and night time gs. In
the first run, measurement were taken on four occasions for diurnal gs and on
seven occasion for nocturnal gs between 25 to 47 DAP, using a cycling leaf
porometer. In the second run, both diurnal and nocturnal gs were taken on four and
five occasions, respectively between 35 to 50 DAP using the cycling leaf
porometer. The same leaf was used for all measurements on each day, avoiding
major veins, and the next youngest leaf as it became fully expanded was used on
subsequent days. An index of stomatal responsiveness to O3 was calculated from
mean gs in each O3 and water treatment (WW or WD), as the stomatal response
from low O3 to high O3 normalized by gs at low O3.
Chlorophyll content of the plants was measured with a young fullyexpanded leaf with the portable chlorophyll meter described earlier on 5 separate
occasions between 25 to 45 DAP in the first run and 3 separate occasions between
30 to 50 DAP in the second run. Plants were destructively harvested at 47 and 56
DAP in the first and second run, respectively. The protocols for separating plants
parts, determination of leaf area, dry weight recording, and calculation of
allometric parameters were similar as that described for Common waterhemp.
67
Projected Water Use
Canopy transpiration in each treatment was simulated from single leaf
measurements as:
T = (VPD) (gs) (LAI)
Eq. 1
Where T is transpirational water loss s-1 m-2 of leaf, VPD is leaf to air vapor
pressure difference, gs is single leaf stomatal conductance, and LAI is leaf area per
unit ground area. VPD was fixed at 0.02 mol mol-1 at night and 0.04 mol mol-1 at
midday, and LAI was fixed at 2 m2 m-2 (Nordby and Hartzler, 2004).
Experimental Design and Data
Analysis
The experimental design for both the species was a randomized complete
block arranged as a split-plot. The main effect was O3 concentration and the subeffect was the irrigation regime. The experiment was replicated in three blocks of
CSTRs aligned parallel to windows and cooling fans in the greenhouse to isolate
location effects. One CSTR in each block was exposed to each of the three O3
concentrations.
Data were analyzed using SAS for Windows (v. 9.2.1) using general linear
model procedure (PROC GLM). Data that failed to meet the normality
assumptions of ANOVA were log-transformed prior to analysis. This was only
necessary for the gs data. Ozone treatments and irrigation sub-treatments were
considered fixed factors and block and runs were considered as random factors.
Interactions between the various factors were also tested. The data for the two
weed species were analyzed separately. For each weed species, the two runs
yielded consistent results as there was no run by treatment interactions (P > 0.05).
Therefore, data for the two runs were pooled for analysis. When the effect of a
68
treatment was significant by F test (P < 0.05), means were separated using
Fisher’s Least Significant Difference (LSD) test.
Results and Discussion
For all parameters tested, except chlorophyll concentration in Palmer
amaranth, the O3 and irrigation interaction was not significant, so that experimentwise effects of O3 were evaluated across the irrigation regimes. In the case of
chlorophyll concentration in Palmer amaranth, there was an interaction between
the O3 concentration and irrigation regime. Therefore, data for irrigation regime in
this parameter was analyzed separately for each O3 concentration.
Stomatal Conductance
In common waterhemp, gs declined throughout the day with lower
maximum values (Fig. 7b). Common waterhemp showed significant amount of
night time gs which was almost half of the day time gs and four fold greater than
the night time gs taken at the same time in cotton (Experiment II). There was no
effect of O3 or irrigation regime in both daytime and night time gs in common
waterhemp (Fig. 7b).
Stomatal conductance in Palmer amaranth showed a bell-shaped diel
pattern during the daytime (Fig. 7a) which was similar to cotton as explained
earlier in Experiment II. However the values of daytime gs in Palmer amaranth
were lower than in cotton and higher than common waterhemp; although, these
were separate studies and direct comparisons cannot be made statistically. Palmer
amaranth showed maximum values of gs during mid-day with decreasing values
during the evening time. Similar to common waterhemp, there was no effect of O3
or irrigation regime on daytime gs. Palmer amaranth also showed significant
amount of night time gs similar to common waterhemp and was also more than
69
four-fold greater than in cotton (Experiment II). The night time gs in Palmer
amaranth showed a consistent decline over the night hours with increasing values
during morning hours (Fig. 7a). Ozone had no effect on the night time gs of Palmer
amaranth. However, WD conditions reduced night time gs in Palmer amaranth but
not in common waterhemp (Fig. 7b).
Canopy transpiration simulated from single leaf measurement in common
waterhemp showed high night time gs which was almost 40% of daytime gs in O3free condition (Table 6). Canopy transpiration simulated from single leaf
measurement in WW plants in Palmer amaranth showed significant night time
transpiration which was almost 53% greater than that in WD condition and more
than half (57%) of mid-day transpiration in WW condition (Table 6).
Stomatal conductance is key to determining the O3 flux (Davison 1998) and
WD stress in plants. Stomata regulate leaf diffusive conductance which results in
water loss and carbon gain (Buckley 2005). In many crops, such as cotton,
daytime gs reduced in high O3 condition (Temple 1986; Temple1990). In contrast
to these results, in this current study, there was no effect on daytime gs of O3 and
WD conditions on both Palmer amaranth and common waterhemp. This might be
due to high amount of variability in stomatal condition in weed species. Becker et.
al. (1989) showed a strong correlation between O3 resistance in white clover
varieties and gs. However, there are very few studies that have looked at the effect
of O3 on gs in weed species.
70
Figure 7. The effect of irrigation regime (averaged over chronic ozone (O3)
exposure) on the diel time course of stomatal conductance (gs) in (a) Common
waterhemp (b) Palmer amaranth. Plants were exposed to well-watered (WW) and
water deficit (WD) condition
Table 6. Impact of ozone and irrigation regime on calculated water consumption of common waterhemp and Palmer
amaranth
Time of
Day
(hrs)
Species
Ozone conc.
(ppb)
Irrigation
regime
Stomatal
Conductance
mmol m-2 s-1
Leaf-Air
VPD
LAI1
Transpiration
Effect on
Transpiration2
mol/mol
m2/m2
mm/hr
mm/month
02:00
02:00
Common waterhemp
Common waterhemp
4
114
WW&WD
WW&WD
55.6
51.5
.02
.02
2
2
0.14
0.13
-1.9
13:30
13:30
Common waterhemp
Common waterhemp
4
114
WW&WD
WW&WD
75.6
58.2
.04
.04
2
2
0.39
0.30
-8.0
1:30
1:30
1:30
1:30
Palmer amaranth
Palmer amaranth
Palmer amaranth
Palmer amaranth
4
4
114
114
WW
WD
WW
WD
54.38
35.32
35.388
29.75
.04
.04
.04
.04
2
2
2
2
0.28
0.18
0.18
0.15
13:30
13:30
Palmer amaranth
Palmer amaranth
4
114
WW&WD
WW&WD
187.62
185.96
.02
.02
2
2
0.48
0.48
-17.60
-5.20
-0.38
1
Source : Nordby and Hartzler,2004
2
Differences between exposure of 4 ppb ozone and 114 ppb O3 assuming 3 h of average midday transpiration plus 6 h of nocturnal
transpiration per day for 30 d in a month.
71
72
Incomplete stomatal closure during night observed in both Palmer amaranth
and common waterhemp was similar to that in other species such as in Pinus
ponderosa (Grulke et al., 2004) and Betula pendulata (Matyssek et al., 1995). The
occurrence of high night time gs can play an important role in uptake of air
pollutants such as O3 (Caird et al., 2007; Musselman and Minnick, 2000).
Although there was high night time gs, this was not affected by O3 exposure levels
as observed in cotton (objective II)
Leaf Parameters
Ozone and irrigation regime had no effect on the chlorophyll concentration
of young fully expanded common waterhemp leaves. There was no interaction of
O3 and irrigation regime either for chlorophyll concentration. Ozone and irrigation
regimes did not affect other leaf parameters such as total number of leaves, total
leaf area, and leaf dry weight either. However, increasing O3 concentrations
reduced leaf mass per area (LMA) (Table 7). Leaf mass area declined substantially
at moderate and high O3 compared to low O3.
An interaction occurred between O3 concentration and irrigation regime for
chlorophyll concentration in Palmer amaranth. Chlorophyll concentration was
greater in WD than in WW conditions under high O3 concentration (Fig. 8).
Similar to common waterhemp, O3 and irrigation regime had no effect on other
leaf parameters such as total number of leaves, leaf area, leaf weight, and LMA
(Table 8). No necrotic lesions were observed in either of the species. Although
necrotic lesions are commonly observed with chronic exposure to high O3
(Goumenaki et al., 2010; Pell et al., 1997; Sarkar et al., 2010), they were not
observed in these Amaranth plants. Ozone had no effect on LMA and root to shoot
ratio either in another weed species, horseweed (Grantz et al., 2008). However, in
horseweed, O3 reduced leaf area and shoot biomass (Grantz et al., 2008).
73
Figure 8. The effect of irrigation regime and chronic ozone (O3) exposure on
Chlorophyll content (SPAD units) (a) Common waterhemp (b) Palmer amaranth
Note : There was no effect of ozone and water level in common waterhemp,
therefore the data for WW and WD is combined. However, In Palmer amaranth,
there was interaction between ozone and water level therefore the data presented
separately for WW and WD.
Table 7. Impact of ozone and irrigation regime on leaf properties of Common waterhemp.
Total leaves2
Leaf area2
Leaf dry wt.2
Leaf mass area2
(leaves plant-1)
(cm² plant-1)
(g plant-1)
(g m-2)
4
94.7a
91.42a
0.53a
64.56a
59
78.5a
93.65a
0.40a
42.07b
114
73.4a
93.69a
0.35a
36.58b
WW
84.40A
99.21A
0.44A
46.61A
WD
77.38A
86.44A
0.41A
48.25A
Ozone
0.5052
0.9909
0.1449
0.0419
Irrigation
0.4699
0.2932
0.6685
0.7500
Treatment
Ozone conc. (ppb)
Irrigation regime1
P-values
Ozone X Irrigation
0.4402
0.1035
0.4997
0.8981
1
-1
-1
-1
-1
WW = well-watered (600 ml pot day ); WD = water deficit (300 ml pot day ).
2
Means within a column for ozone concentration followed by the same lowercase letters and the same uppercase
letters for the irrigation regime are not significantly different at a 0.05 level of significance according to Fisher’s
Least Significant Difference (LSD) test
74
Table 8. Impact of ozone and irrigation regime on leaf properties of Palmer amaranth.
Total leaves2
Leaf area2
Leaf dry wt. 2
Leaf mass area2
(leaves plant-1)
(cm² plant-1)
(g plant-1)
(g m-2)
4
133a
972.09a
3.63a
37.57a
59
141a
903.77a
3.35a
37.21a
114
131a
887.42a
3.2a
36.08a
WW
129A
872.35A
3.31A
37.67A
WD
141A
969.70A
3.49A
36.16A
Ozone
0.7445
0.2483
0.5941
0.9155
Irrigation
0.5857
0.3485
0.7390
0.2360
Treatment
Ozone conc. (ppb)
Irrigation regime1
P-values
Ozone X Irrigation
0.9438
0.8258
0.9612
0.3334
1
WW = well-watered; WD = water deficit.
2
Means within a column for ozone concentration followed by the same lowercase letters and the same uppercase
letters for the irrigation regime are not significantly different at a 0.05 level of significance according to Fisher’s
Least Significant Difference (LSD) test
75
76
Shoot Parameters
Ozone and irrigation regime had no effect on aboveground plant
parameters such as plant height, stem dry weight, leaf insertion rate, and shoot dry
weight in neither common waterhemp (Table 9) nor Palmer amaranth (Table 10).
Ozone has generally been reported to reduce shoot biomass in several plant
species (Heggestad et al., 1988; McLaughlin et al., 1982; Miller, 1988; Pell et al.,
1994). However, increasing levels of O3 was reported to have no effect on both
shoot biomass and root biomass of weed species such as yellow nutsedge (Grantz
and Shrestha, 2005). Therefore, these Amaranth species seem to be tolerant to
both O3 and moisture stress levels tested in this study.
Root Parameters
Similar to the shoot parameters, O3 and irrigation regimes had no effect on
root productivity in either of the Amaranthus species (Tables 11, 12). There was
no interaction between O3 and irrigation regime on root parameters for either of
the species. . Two allometric parameters were derived to further assess the effect
of O3 and irrigation regime on plant parameters in both Palmer amaranth and
Common waterhemp i.e., root to shoot biomass ratio and root biomass per leaf
area. The root to shoot ratio was similar between the plants under the two
irrigation regimes and there was no interaction between the O3 and irrigation
regime in both the species (Table 11, 12). Ozone had no effect on root to shoot
ratio of both the species. Although O3 had no effect on the root mass per leaf area
in Palmer amaranth, the intermediate O3 concentration reduced the root mass per
leaf area in common waterhemp (Table 11). However, there was no effect of O3 or
irrigation regime on root mass per area of Palmer amaranth (Table 12). Contrary
to these results, Grantz et al., (2008) reported reductions in root biomass of
horseweed under increasing O3 concentrations. However, similar to this study,
Table 9. Impact of ozone and irrigation regime on shoot parameters of Common waterhemp.
Plant height2
Stem dry wt.2
(cm)
(g plant-1)
4
77.73a
1.87a
1.15a
2.40a
59
73.48a
1.68a
1.05a
2.08a
114
72.11a
1.49a
1.13a
1.85a
WW
76.91A
1.73A
1.09A
2.18A
WD
71.75A
1.64A
1.13A
2.05A
Ozone
0.8066
0.6291
0.6520
0.4829
Irrigation
0.3278
0.7622
0.7533
0.7041
Treatment
Leaf insertion rate2
Shoot dry wt. 2
(g plant-1)
Ozone conc. (ppb)
Irrigation regime1
P-values
Ozone X Irrigation
0.9896
0.5946
0.1813
0.5345
1
WW = well-watered (600 ml pot-1 day-1); WD = water deficit (300 ml pot-1 day-1).
2
Means within a column for ozone concentration followed by the same lowercase letters and the same uppercase
letters for the irrigation regime are not significantly different at a 0.05 level of significance according to Fisher’s
Least Significant Difference (LSD) test.
77
Table 10. Impact of ozone and irrigation regime on shoot parameters of Palmer amaranth.
Plant height 2
Stem wt.2
Leaf insertion rate2
Shoot wt. 2
(cm)
(g plant-1)
(leaves cm-1)
(g plant-2)
4
64.45a
4.59a
2.10a
7.82a
59
71.58a
4.53a
1.94a
7.88a
114
64.75a
4.18a
2.04a
7.41a
WW
67.13A
4.67A
2.13A
7.98A
WD
66.85A
4.17A
1.93A
7.42A
Ozone
0.4913
0.7470
0.2110
0.8352
Irrigation
0.9478
0.2444
0.5236
0.4927
Treatment
Ozone conc. (ppb)
Irrigation regime1
P-values
Ozone X Irrigation
0.4630
0.7436
0.9575
0.8482
1
WW = well-watered; WD = water deficit.
2
Means within a column for ozone concentration followed by the same lowercase letters and the same uppercase
letters for the irrigation regime are not significantly different at a 0.05 level of significance according to Fisher’s
Least Significant Difference (LSD) test
78
Table 11. Impact of ozone and irrigation regime on root parameters of Common waterhemp.
Treatment
Root dry wt.2
Total biomass2
Root Shoot Ratio2
Root mass per leaf
area 2
(g plant-1)
(g plant-1)
(g plant-2)
4
0.46a
2.87a
0.20a
62.99a
59
0.35a
2.44a
0.18a
37.73b
114
0.37a
2.22a
0.20a
46.85ab
WW
0.41A
2.59A
0.20A
50.51A
WD
0.38A
2.43A
0.20A
47.28A
Ozone
0.4936
0.463
0.7288
0.0419
Irrigation
0.7083
0.6853
0.8729
0.6288
Ozone conc. (ppb)
Irrigation regime1
P-values
79
Ozone X Irrigation
0.8638
0.5973
0.8198
0.0821
1
WW = well-watered (600 ml pot-1 day-1); WD = water deficit (300 ml pot-1 day-1).
2
Means within a column for ozone concentration followed by the same lowercase letters and the same uppercase
letters for the irrigation regime are not significantly different at a 0.05 level of significance according to Fisher’s
Least Significant Difference (LSD) test.
Table 12. Impact of ozone and irrigation regime on root parameters of Palmer amaranth.
Treatment
Root wt.2
Total biomass2
Root Shoot Ratio2
Root mass per
leaf area 2
(g plant-1)
(g plant-1)
(g plant-2)
4
1.69a
9.52a
0.21a
18.91a
59
1.60a
9.48a
0.21a
18.7a
114
1.59a
9.00a
0.21a
18.34a
WW
1.67A
9.02A
0.21A
18.09A
WD
1.59A
9.65A
0.21A
19.17A
Ozone
0.8638
0.8556
0.8716
0.9838
Irrigation
0.7239
0.5141
0.6662
0.5229
Ozone conc. (ppb)
Irrigation regime2
P-values
Ozone X Irrigation
0.8920
0.9127
0.9191
0.7884
1
WW = well-watered; WD = water deficit.
2
Means within a column for ozone concentration followed by the same lowercase letters and the same uppercase
letters for the irrigation regime are not significantly different at a 0.05 level of significance according to Fisher’s
Least Significant Difference (LSD) test
80
81
increasing levels of O3 had no effect on root biomass of yellow nutsedge (Shrestha
and Grantz, 2005). Thus, these amaranth species seem tolerant to both O3 and
water stress and may be more competitive than weed species such as horseweed.
Conclusion
Although there is increasing interest in effects of O3 and regulated deficit
irrigation in crop species and many studies have been conducted on several crops,
very few studies have been conducted on weed species. Weeds play a significant
role in agroecosystems and considerable amount of money is spent on weed
management. Therefore, it is important to study the response of weed species to
these environmental factors. This study determined that increased O3
concentrations and the level of water stress tested had no adverse effect on either
the shoot or root parameters of these amaranth species. Both these species, thus,
may have considerable tolerance to increased O3 concentrations and decreased
moisture levels, both of which are common environmental conditions in the SJV.
Furthermore, stomatal conductance is key in determining the effect of many
stresses such as O3 stress and water deficit in plants. Although the effects of these
stresses on stomatal conductance have been studied in several crop and forest tree
species, very few studies have been conducted on weed species. This study was
the first of its kind to study stomatal response under O3 and water stress in two
important weed species in a 24 h period. Both Palmer amaranth and common
waterhemp had high nocturnal stomatal conductance and high night time
transpiration. These factors could result in nigh-time water loss and affect water
availability for crops and reduce irrigation efficiency. Therefore, these amaranth
species may proliferate and become an even more serious pest in cropping systems
in future scenarios of climate change.
82
Furthermore, they may even out compete some other weed species that are
not as tolerant to O3 and moisture stress. However, the effect of these factors on
the fecundity of these two amaranth species is not known because the plants were
not grown until maturity. The resistance of weed species to O3 could also
accelerate due to their short life cycles and prolific reproduction (Grantz et al.,
2005). The effect of these factors on fecundity needs to be evaluated to predict
their population dynamics under these two environmental conditions.
Nevertheless, this study demonstrated the invasive potential of common
waterhemp and Palmer amaranth under current and future climatic scenarios in the
San Joaquin Valley of California.
83
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