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The effect of elevated CO2 and water management on photosynthesis and photosynthate partitioning of rice leaves

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The effect of elevated CO2 and water management on photosynthesis and photosynthate partitioning of rice leaves
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Widodo, 1958-
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English
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xi, 107 leaves : ; 29 cm.

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Rice -- Effect of carbon dioxide on ( lcsh )
Photosynthesis ( lcsh )
Carbon dioxide -- Environmental aspects ( lcsh )
Agronomy thesis, Ph. D ( lcsh )
Dissertations, Academic -- Agronomy -- UF ( lcsh )
Carbon dioxide -- Environmental aspects. ( fast )
Photosynthesis. ( fast )
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bibliography ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis (Ph. D.)--University of Florida, 1996.
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Includes bibliographical references (leaves 91-106).
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Typescript.
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Vita.
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by Widodo.

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University of Florida
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University of Florida
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Full Text
THE EFFECT OF ELEVATED CO2 AND WATER DEFICIT ON
PHOTOSYNTHESIS AND PHOTOSYNTHATE PARTITIONING OF RICE LEAVES
By
WIDODO
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1996




ACKNOWLEDGMENTS
I wish to express my sincere appreciation to committee members Dr. George E. Bowes, Dr. Cu V. Vu, Dr. Leon H. Allen Jr., Dr. Raymond N. Gallaher and Dr. Kenneth J. Boote. The direction and support they have provided during my doctoral studies has been invaluable. I would like to give special thanks to my committee chairman and advisor, Dr. Kenneth J. Boote, for his numerous ideas, patience, and guidance throughout my research.
I would like further to thank Drs. L.H. Allen Jr., K.J. Boote, and C.V. Vu for providing the oportunity to carry out the CO2 enrichment study on rice and for providing all the necessary facilities for carbohydrates and enzyme analyses. I would like to extend my appreciation to Dr. J.T. Baker for conducting whole-canopy chamber experiments, and for his suggestions and help. Jean Marie Thomas, Joan Anderson and Pan Deyun deserve many thanks. They offered invaluable help and guidance for my laboratory and field studies.
I thank my parents who taught me the values needed to complete this degree. I also thank my children, Okky and Intan, whose cheerfulness gave added incentive to complete my studies. The greatest thanks go to my wife Pratiwi
ii




Widiastuti, whose patience, hard work, and understanding allowed me to accomplish my degree.
Finally, I would like to acknowledge the Higher Education Development Projects of Indonesian Government for providing scholarship and financial assistance under the USAID program.
iii




TABLE OF CONTENTS
ACKNOWLEDGMENTS.................... . ... . .. .. . ...
LIST OF TABLES............................vi
LIST OF FIGURES........................ix
ABSTRACT...............................x
CHAPTERS
1 LITERATURE REVIEW.........................1
Introduction............................
Effects of CO2 on Photosynthetic Enzymes 4
Effects CO2 and Drought Stress on Soluble
Protein and chlorophyll ..................6
Effects of CO2 and Water Stress on
Sucrose Synthesis Enzymes ......... 7
Effects of CO2 and Water Stress on
Photosynthesis...................10
Ef fects of CO2 and Water Stress on Non-Structural
Carbohydrates......................20
2 EFFECTS OF ELEVATED CO2 CONCENTRATION AND WATER DEFICIT
ON RICE LEAF PHOTOSYNTHESIS, CHLOROPHYLL AND SOLUBLE PROTEIN.............................28
Introduction.......................28
Materials and Methods..............31
Results and Discussion...............35
3 RICE LEAF NON-STRUCTURAL CARBOHYDRATES IN RESPONSE TO
ENHANCED CO2 AND WATER STRESS............50
Introduction........................50
Materials and Methods..............53
Results and Discussion...............55
4 EFFECTS OF ELEVATED CO2 CONCENTRATION AND WATER STRESS ON SUCROSE PHOSPHATE SYNTHASE ACTIVITY . 71
Introduction........................71
iv




Materials and Methods . . . . . . 74
Results and Discussion . . . . . . 75
5 COORDINATION OF CARBON METABOLISM ACTIVITIES . 81 6 SUMMARY AND CONCLUSIONS . . . . . . 87
REFERENCE LIST 91
BIOGRAPHICAL SKETCH 107
v




LIST OF TABLES
Table page
2.1 Times when water was withheld and restored for
drought treatments for all chambers. Rice was planted
on 15 July 1994 34
2.2 Leaf photosynthetic rate of rice plants grown at
350 and 700 jjL C02 L 1 and in four different
water managements. Measurements were taken on 51, 55, and 60 day-old plants (before stress affected)
and 72 day-old plants (early water deficit
period). 42
2.3 Leaf photosynthetic rate of rice plants grown at
350 and 700 jiL C02 L-1 and in four different
water managements. Measurements were taken at noon
eastern standard time from 74 (stressed), 75
(stressed), 82 and 84 (recovery) day-old plants. 43
2.4 Leaf photosynthetic rate of rice plants grown at
350 and 700 pL C02 L-' and in four different
water managements. Measurements were taken on 89,
98 and 100 day-old plants 44
2.5 Leaf photosynthetic rate of rice plants grown at
350 and 700 jiL C02 L-1 and in four different
water managements. Measurements were taken from
109, 114 and 125 day-old plants . . . . . 45
2.6 Leaf soluble protein concentration of rice plants
grown at 350 and 700 pL C02 L and in four
different water managements. Measurements were
taken from 54, 74, 82 and 92 day-old plants . . 46
2.7 Leaf soluble protein concentration of rice plants
grown at 350 and 700 PL C02 L-1 and in four
different water managements. Measurements were
taken from 100, 110 and 127 day-old plants . . 47
2.8 Leaf chlorophyll concentration of rice plants
grown at 350 and 700 pL C02 L-1 and in four
different water managements. Leaf samples were
taken from 54, 74, 82 and 92 day-old plants . . 48
vi




2.9 Leaf chlorophyll concentration of rice plants
grown at 350 and 700 p.L CO2 L 1 and in four
different water managements. Measurements were
taken from 100, 110 and 127 day-old plants . . 49
3.1 Leaf sucrose concentration of rice plants grown
at 350 and 700 IlL CO2 L-1 and in four different water managements. Leaf samples were taken at
noon eastern standard time from 55, 58, 72 and
74 day-old plants...................62
3.2 Leaf sucrose concentration of rice plants grown
at 350 and 700 ilL CO2 L-1 and in four different water managements. Leaf samples were taken at noon eastern standard time from 75 (stressed),
82 and 89 (recovery) day-old plants..........63
3.3. Leaf sucrose concentration of rice plants
grown at 350 and 700 IL CO2 L 1 and in four
different water managements. Leaf samples were
taken at noon eastern standard time from 100,
109 (deficit period) and 125 (recovery period)
day-old plants.....................64
3.4. Leaf starch concentration of rice plants
grown at 350 and 700 iiL CO2 L-1 and in four
different water managements. Leaf samples were
taken at noon eastern standard time from 55, 58,
72 and 74 day-old plants ...............65
3.5 Leaf starch concentration of rice plants grown
at 350 and 700 IlL CO2 L-1 and in four different water managements. Leaf samples were taken at
noon eastern standard time from 75 (stressed),
82 and 89 (recovery) day-old plants..........66
3.6 Leaf starch concentration of rice plants grown
at 350 and 700 liL CO2 L-1 and in four different water managements. Leaf samples were taken at
noon eastern standard time from 100, 109
(stressed) and 125 (recovery) day-old plants . . 67
3.7. Leaf fructose concentration of rice plants grown
at 350 and 700 IlL CO2 L-' and in four different water managements. Leaf samples were taken at
noon eastern standard time from 55, 58, 72
and 74 day-old plants ....... ...........68
3.8 Leaf fructose concentration of rice plants grown
at 350 and 700 IlL CO2 L-1 and in four different water managements. Leaf samples were taken at noon eastern standard time from 75 (stressed),
vii




82 and 89 (recovery) day-old plants .. ........ ..69
3.9 Leaf fructose concentration of rice plants grown
at 350 and 700 UL C02 L-1 and in four different water managements. Leaf samples were taken at
noon eastern standard time from 100, 109
(stressed) and 125 (recovery) day-old plants . . 70
4.1 Sucrose phosphate synthase activities of rice
plants grown at 350 and 700 IiL C02 L 1 and in
four different water managements. Plant samples
were taken at noon eastern standard time from 54,
74 (stressed), 82 and 92 (recovery) day-old
plants .......... ....................... .79
4.2 Sucrose phosphate synthase activities of rice
plants grown at 350 and 700 IiL C02 L and in
four different water managements. Plant samples
were taken at noon eastern standard time from
100, 110(stressed) and 127 (recovery)
day-old plants .. ............. .. ......... ..80
viii




LIST OF FIGURES
Figure page
5.1. Time course of sucrose and starch concentration and
SPS activity under continuously-flooded and waterdeficit at panicle initiation treatments ..... .. 83
5.2. Time course of sucrose and starch concentration and
SPS activity under water deficit at anthesis and at
both phase treatments .... .............. 84
ix




Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy
THE EFFECT OF ELEVATED CO2 AND WATER DEFICIT ON
PHOTOSYNTHESIS AND PHOTOSYNTHATE PARTITIONING OF RICE LEAVES By
Widodo,
December 1996
Chairperson: Kenneth J. Boote
Major Department: Agronomy
The carbon dioxide concentration of the earth's atmosphere and frequency and intensity of rainfall are
predicted to change in future years. The resulting changes could have considerable impact on crop production. The
objective of this study was to evaluate responses of rice leaves to CO2 concentration and water management for rice grown in controlled environmental chambers. Leaves of rice plants responded differently to CO2 concentration and various water managements. High-CO2 concentration significantly increased leaf photosynthetic rate during the study. On several dates, leaf soluble protein responded negatively by decreasing under increased CO2 concentration. Nevertheless, leaf chlorophyll concentration of leaves of high-CO2 treatments was significantly higher compared to that of lowCO2 treatments. High-CO2 treatments significantly increased
x




leaf sucrose, starch and fructose concentration by 0.0 to 25.8%, 0 to 40.9% and 12.2 to 64.0%, respectively. High-CO2 concentration had significant enhancing effects on sucrose and starch accumulation during early reproductive phases, but not during later reproductive phases. The difference in leaf
starch, sucrose, and fructose concentration between leaves grown at elevated and current CO2 diminished with plant maturity. High-CO2 concentration also increased SPS activity throughout the season.
Water stress treatment significantly affected a number of variables during panicle initiation and anthesis stages. water stress caused major reductions in leaf photosynthetic
rate, leaf chlorophyll, and soluble protein, and water deficit periods also caused major decreases in leaf sucrose, starch and fructose concentration, and also in SPS activity. Water stress had more profound effects on leaves of plants grown in
low-CO2 concentration. High-CO2 plants were able to maintain leaf photosynthesis longer into the water deficit period and had smaller reductions in chlorophyll and fructose concentration compared to ambient CO2 plants.
xi




CHAPTER 1
LITERATURE REVIEW
Introduction
The amount of carbon dioxide (C02) in the earth's
atmosphere has risen over the last century because of human activities that release CO2 from terrestrial reservoirs
(Roeckner, 1992). These activities include energy production and use, manufacturing and agricultural processes, land use changes, and waste disposal (Subak et al., 1993). Over the past four decades, observations of atmospheric CO2 concentration ([CO2]) at Mauna Loa, Hawaii, and at the South Pole show an approximate proportionality between the rising atmospheric concentrations and industrial CO2 emissions (Keeling et al., 1995). During the past 200 years, the atmospheric [CO2] has increased from 280 ppmv to 360 ppmv. Atmospheric [C02] is expected to reach twice the preindustrial level by the middle of the next century (Keeling et al., 1995). Ice core analyses of polar ice has revealed a high correlation between climatic change and variations in the atmospheric concentrations of greenhouse gases (CO2 and
methane) over the last 160,000 years (Roeckner, 1992).
When greenhouse gas concentration increases, atmospheric moisture variability is substantially larger over areas that
1




2
experience the greatest surface warming (Liang et al., 1995). Results from a coupled biosphere-atmosphere model (SiB2-GCM) indicate that, for doubled CO2 conditions, evapotranspiration will decrease and air temperature will increase over the tropical continents, amplifying the changes resulting from atmospheric radiative effects (Sellers et al., 1996). Moreover, the global mean surface temperature will rise between 1.5 0C and 4.5 C (Houghton et al., 1995). In
addition, under double CO2 condition, variability of daily precipitation will increase. There will be some areas with increases and decreases of frequency and intensity of
precipitation (Mearns et al., 1995). The resulting precipitation shifts could have considerable agricultural impact especially in regions that become drier (Wigley et al., 1980). Since soil water availability currently limits crop growth more than all other environmental factors combined (Boyer, 1982), it is important to investigate the effects and possible interactions of water regimes with [C021 upon major food crops such as rice.
Rice (Oryza sativa L.) provides over half the diet of 1.6 billion people and is the only grain crop used almost exclusively for human food consumption. World rice production is second after wheat production. Rice is grown over a wide range of latitudes from roughly 53' N to 40' S and to elevations of more than 2500 m (Mooreman and van Breeman, 1978) Of the total of 140 to 145 million hectares of land




3
area planted to rice annually (IRRI, 1982), nearly 12% of the world's rice area is devoted to upland rice culture. Upland rice yields are lower than lowland rice, mainly due to complete dependence on rainfall for moisture (De Datta and Beachell, 1972). Moreover, about half of the world's rice land area depends on rainfall and is often subjected to drought stress (IRRI, 1982).
Several researchers have conducted studies of the effects of rising [C021 on rice (Yoshida, 1976; Baker et al., 1990; Baker et al., 1992; Ziska and Teramura, 1992a, 1992b; Ziska et al., 1996). In response to superambient Ca2, rice photosynthesis, biomass, grain yield, and leaf carbohydrates increased up to about 500 pbar CO2 (Yoshida, 1976); but above this, gains were minimal (Baker et al., 1990; Baker et al., 1992). The photosynthetic and growth response to elevated C02 may be highly dependent on the supply of N (Ziska et al., 1996).
The availability of water is one of the most important factors determining vegetation diversity and plant productivity (Rochefort and Woodward, 1992). The effects of water deficits on plant performance and growth are mediated through decreases in stomatal conductance and photosynthesis and depend on the severity and duration of the drought period, the presence of further environmental constraints, and species inherent characteristics (Chaves and Pereira, 1992). water stress can have profound metabolic effects in plants,




4
resulting not only in impaired gas exchange but also in profound alteration of physiological processes, such as cell growth, wall synthesis, nitrogen and chlorophyll metabolism, and the levels of growth substances (Hsiao, 1973).
Rice is most sensitive to water stress around the flowering stage (Yoshida, 1978). Moisture stress at anthesis results in the failure of the panicle to exert fully from the flag leaf sheath. Reduced panicle exertion was shown to be regulated by the plant water status. A direct cause and effect relationship was also noted between panicle exertion and spikelet fertility (O'Toole and Namuco, 1983). In the rice plant, 60-90% of the total carbon in panicles is derived from photosynthesis after heading, and the flag leaf is the organ that contributes the most to grain filling (Yoshida, 1981). Under water stress (Boyer, 1976; Kreidemann and Downton, 1981; Sinha et al., 1982; Yoshida, 1978) and (Clifford et al., 1993; Jones, 1993; Yoshida, 1976) C02 enrichment induces changes in crop growth, metabolites and enzymes associated with assimilatory and degradative reactions in plants.
Effect of CO on Photosynthetic Enzymes
The enzyme responsible for initiating C3 photosynthesis, rubisco, has been the focus of much attention regarding the regulation of the rate of carbon entering the photosynthetic pathway. Rubisco, ribulose-l,5-bisphosphate (RuBP)
carboxylase/oxygenase (Rubisco), catalyses two different




5
reactions in the chloroplast stroma (Campbell et. al., 1988). This enzyme acts as carboxylase with the substrates RuBP and CO2 and as an oxygenase with the substrates RuBP and 02. The former reaction results in CO2 uptake of photosynthesis whereas the latter results in mitochondrial CO2 evolution called photorespiration. Thus the net rate of assimilation of CO2 in a leaf depends on the rates of RuBP carboxylation and RuBP oxygenation. The ratio of carboxylation to oxygenation is determined by the concentration of the substrates CO2 and 02, and by the catalytic properties of the enzyme with respect to these substrates (Brooks and Farquhar, 1985). The dependence of Rubisco activity (carbamylation) on PFD can be altered by CO2, 02, and temperature. For example, under atmospheric conditions (21% 02 and 350 ppm CO2) and normal temperature, rubisco activity generally varies with PFD by only two to three fold. However, at 2% 02 and below, a reduction of greater than 90% of maximal Rubisco activity can be observed at very low PFD. In some cases, especially under high CO2 and low temperatures (140C), the changes in Rubisco activity appear to be correlated with a reduced ATP/ADP ratio associated with a reduction in photosynthesis by a "feedback effect" (Portis, 1992). In intact C3 plants, increasing temperature causes an increase in both the CO2 compensation point and the inhibition of net photosynthesis by 02. This suggests that the rate of photorespiration relative to the




6
rate of photosynthesis increases with temperature (Machler and Nosberger, 1980).
Effect of CO and Drought Stress on
Soluble Protein and Chlorophyll
Schwanz et al. (1996) reported that antioxidants, protective enzymes, soluble protein, and pigments showed considerable fluctuations in a two year experiment on oak (Quercus robur) and pine (Pinus pinaster) seedlings in response to enhanced [CO2] and drought stress. When the seedlings were subjected to drought stress by withholding water, the activities of antioxidative enzymes decreased in leaves of pine and oak grown at ambient [Ca2] and increased in plants grown at elevated [CO2]. The authors suggested that growth in elevated CO2 might reduce oxidative stress to which leaf tissues are normally exposed and enhance metabolic flexibility to encounter increased stress by increases in antioxidative capacity. Ficus opuntia (Jacob et al., 1995) grown at elevated [Ca2] also contained less soluble protein (39-52%). The rubisco content was 43 to 58% of soluble protein. Leaf chlorophyll content per unit area or dry mass was significantly lower in elevated C02-grown plants and increased significantly with increasing nutrient availability (Radoglou and Jarvis, 1992). Hunt et al. (1996) found that after two growing season, plant tissue N concentrations of C3 and C4 grasses were lower under elevated C02.




7
Effects of CO and Water Stress on Sucrose Synthesis Enzymes
When stomata close in response to water stress, CO2 concentration inside the leaf goes down, ultimately leading to deactivation of sucrose phosphate synthase (SPS) activity. This decrease in SPS activity shows up in measurements of gas exchange under water stress even though it may be a
consequence of low CO2 (Vassey et al., 1991). When water stress occurs for several days, the reduction in SPS capacity is no longer reversed by a 20 min incubation in high CO2 but requires 5 h for recovery. These results indicate that the activity of SPS can be influenced by the CO2 concentration surrounding the plant (Vassey et al., 1991).
Sucrose Phosphate Synthase
Sucrose Phosphate Synthase (SPS) plays an important role as the limiting factor of partitioning of carbon sources. Therefore, it seems that enhancement of this activity may promote the ability of the source function (Stitt and Quick, 1989). Using isolated maize SPS cDNA which is over-expressed using the promoter of the Rubisco small subunit gene in transgenic tomato, SPS activity was increased six-fold in leaves, resulting in doubling of sucrose content but significant reduction of starch in these leaves (Worrell et al. 1991).




8
SPS is allosterically activated by the presence of glucose-6-phosphate, but inhibited by increase of inorganic phosphate, Pi (Stitt and Quick, 1989). In addition, there is a covalent modification by phosphorylation/dephosphorylation, which regulates its enzyme activity (Walker and Huber, 1989; Huber and Huber, 1992). This modification occurs in response to light and darkness, showing circadian rhythms. In leaves, illumination induces dephosphorylation which leads to the enzyme being activated, and darkness results in inactivation because of phosphorylation (Kalt-Torres et al., 1986; Stitt et al., 1988; Huber and Huber, 1991).
Sakamoto et al. (1995) suggested that function and promoter of rice SPS gene is weak and no typical promoter sequences were found upstream of the coding region. The deduced amino acid sequence of the rice SPS showed a high degree of homology to the known ones. Expression of this gene was detected only in the leaves, suggesting that this gene is specifically expressed in the source organs. Level of expression was extremely low, reflecting weakness of its promoter activity.
After panicle removal at anthesis, Nakano et al. (1995) found that activity of SPS remained relatively high during leaf senescence and decreased more slowly than that in the control plants. Vassey et al. (1991) reported that SPS activity was low in plants held in low CO2 for 1 h. The low-CO2 inhibition of SPS activity could be reversed by incubation of




9
the leaf tissue in high CO2 concentration and high light for 20 min. The activity of soybean SPS was unchanged by long-term C02-enrichment (Huber et al., 1984), but it increased in rice (Hussain et al., 1992). Vassey et al. (1991) concluded that the C02 effect on SPS activity was mediated by the effect of CO2 on the rate of photosynthesis.
Micallef et al. (1995) reported that tomato leaves of SPS-transformed lines were significantly greater (up to 12 times) in limiting and maximum SPS activities. Partitioning of carbon into sucrose increased 50% for the SPS transformants. Intact leaves of the control plants exhibited CO2insensitivity of photosynthesis at high CO2 levels, whereas the SPS transformants did not exhibit C02-insensitivity. Growth at 65 Pa CO2 resulted in reduced photosynthetic capacity for control plants but not for SPS-transformed plants. When grown at 65 Pa CO2, SPS-transformed plants had a 20% greater photosynthetic rate than controls when measured at 65 Pa CO2 and 35% greater rate when measured at 105 Pa CO2. Furthermore, transgenic tomato plants expressing high levels of maize SPS have been concluded to support the postulate that SPS activity can influence the partitioning of carbon between starch and sucrose (Huber and Huber, 1992). In addition, SPS of maize leaves was found in both the mesophyll and bundle sheath cells (Huber et al., 1987).
High [C02] increased leaf blade elongation rate (LER) of expanding blades and in vivo activity (VlimitLng) SPS activity of




10
expanded blades during the early vegetative phase of rice (21 d after planting [DAP]), when tillers were becoming strong carbohydrate sinks. There was a distinct diurnal pattern in LER, SPS activity, and concentration of soluble sugar, with an increase in the early part of the light period and a decrease later in the light period. The higher SPS activity at elevated CO2 at 21 DAP was caused by an increase in the activation state of the enzyme rather than an increase in Vmax. By the mid-tillering stage (42 DAP), CO2 enrichment enhanced SPS activities of source blades (Seneweera et al., 1995).
Effects of CO and Water Stress on Photosynthesis Effects of CO, on Photosynthesis
Many studies have shown that several growth parameters, including leaf area and dry weight, are enhanced during long term exposure to high C02 (Wong, 1979). However, after the initial stimulation of net photosynthetic rate per unit of leaf area by CO2, enhancement may decrease during the
subsequent exposure to high CO2 and a subsequent suppression of photosynthesis may occur (Yelle et al., 1989). The direct effects of [CO2] enrichment on rice (Oryza sativa, L.), soybean (Glycine max, L.) and citrus (various species) was always an increase in photosynthetic rate (Baker and Allen, 1993). Furthermore, photosynthetic rate of rice measured at ambient [C02] decreased with increasing long-term [CO2] growth




treatment due to a corresponding decline in RuBP carboxylase content and activity.
Prolonged exposure to high CO2 leads to changes in biochemical, physiological or morphological factors which may remove or offset the initial stimulation of photosynthesis (DeLucia et al., 1985). Results of experiments with CO2 upon photosynthesis vary with the species investigated.
Nevertheless, by manipulating the CO2 concentration, CO2 can be used to probe the responses of various photosynthetic parameters and to aid in determining their role in regulation of photosynthesis (Kramer, 1981). However, it has been established that acclimation to elevated CO2 alone may lead to a subsequent decline in photosynthetic rate, for which a variety of causes have been invoked, some related to aspect of leaf structure and functioning (Rogers et al., 1983; Sage et al., 1988). However, Idso and Kimball (1991) reported that after a full 3-year period, C02-enriched sour orange trees grown under irrigation and high fertility in the desert environment at Phoenix, Arizona, USA, had consistently sequestered approximately 2.8 times more carbon than the control trees. The authors suggested that these plants may not experience the downward regulation of photosynthetic capacity under field conditions in the natural environment, compared to long term CO2 enrichment plants grown in pots.
On the other hand, Allen and Amthor (1985) pointed out that both the C02-enriched and the nonenriched sour orange




12
trees exhibited midday (and afternoon) depression of photosynthesis under the hot summertime conditions of Phoenix, Arizona. Furthermore, the depression was much more severe in the nonenriched trees than the enriched trees, with leaf photosynthetic rates for the nonenriched trees being only about 1 Umol CO2 m2 s for the last half of the day.
Apparently, elevated CO2 provided protection against midday depression of photosynthesis by some unknown mechanism not related to downregulation.
Ziska and Teramura (1992b) revealed that two rice cultivars (IR-36 and Fujiyama-5) increased 50% in photosynthetic rate when exposed to enhanced [CO2] (660 ibar) and photosynthetic enhancement was still evident after 3 months of exposure to a high CO2 environment. However, in plants exposed to simultaneous increases in CO2 and ultraviolet-B (UV-B) radiation, CO2 enhancement effects on respiration, photosynthesis, and biomass were eliminated in IR-36 and significantly reduced in Fujiyama-5 (Ziska and Teramura, 1992a).
Balaguer et al. (1995) concluded that doubling the atmospheric concentration of CO2 enhanced the rate of net CO2 assimilation by 47%. Zhang and Nobel (1996) studied a C3 desert shrub (Encelia farinosa) and found an increase in assimilation rate by 46% in the early morning, 26% at midday, and 15% in the late afternoon. Balaguer et al. (1995) also found that doubling [CO2] reduced the proportion of fixed




13
carbon retained in the leaf blade, increasing the rate of export. The favorable carbon balance of CO2 enriched leaves was further enhanced by a decrease in the cost of maintenance respiration.
Jacob et al. (1995) reported that Scirpus olneyi grown at elevated CO2 had a significantly higher (30-59%) net CO2 assimilation rate than plants grown at ambient CO2 when measurements were performed on excised stems at the respective growth [CO2]. However, when measurements were made at normal ambient [CO2], net CO2 assimilation rate was smaller (45-53%) in plants grown at elevated [C021 than in those grown at ambient [C02]. Changes in carboxylation efficiency and in situ carboxylase activity were caused by a decreased rubisco concentration (30-58%) in plants grown at elevated [C02].
Van Oosten and Besford (1995) revealed that thylakoid proteins (photosystem I core protein, D, and D2 of the photosystem II core complex, cytochrome f) were all decreased by elevated C02 after 31 days exposure on the fully mature leaves of tomato plants, whereas the large and small subunits of rubisco and Rubisco activase proteins had already declined after 22 d exposure.
Ziska et al. (1996) reported that rice photosynthesis was initially stimulated at the leaf and canopy level with elevated CO2 regardless of supplemental N supply, but with time the photosynthetic response became highly dependent on




14
the level of supplemental N, increasing proportionally as N availability increased.
Baxter et al. (1995) reported that photosynthetic capacity of Poa alpina was reduced by growth at 680 1mol mol1 C02 after 105 d, and that of Fectusa vivipara L. was reduced at 65 d and 189 d after CO2 enrichment began, suggesting downregulation or acclimation. In F. vivipara the relationship between leaf photosynthetic capacity and leaf carbohydrate concentration was such that there was a strong positive correlation between photosynthetic capacity and total leaf N concentration (expressed on a per unit structural dry weight basis), and total nitrogen concentration of successive mature leaves decreased with time.
Atmospheric CO2 enrichment is typically associated with increased rates of leaf photosynthesis and total plant dry matter accumulation. However, increased photosynthetic rates per unit leaf area may not persist for long periods at high atmospheric CO2 concentration (Clough et al., 1980). Ho (1977) reported increased rates of photosynthesis and carbon transport in leaves of tomato plants grown under C02
enrichment, compared to plants grown at ambient CO2.
Barnes et al. (1995) reported that the increase in photosynthesis of Norway spruce (Picea abies [L.1 Karst) induced by C02 enrichment was associated with increased foliar concentrations of glucose, fructose and starch (but no change in sucrose) in the new growth.




15
Maximum carboxylation rates per unit leaf area (Vcmax) were lower in cotton leaves grown at two elevated CO2 concentrations, compared with ambient C02 concentration, under all phosphorus and pot size treatments, indicating that acclimation of photosynthesis had occurred (Barrett and
Gifford, 1995). The degree of photosynthetic acclimation to elevated C02 was not related to the degree by which whole plant carbon gain was stimulated by elevated [CO2] at the different P supplies, or to the degree by which allocation to root and shoots was altered by pot size. Thus there was no simple relationship between photosynthetic and growth acclimation by cotton to elevated CO2. At ambient C02, the maximum carboxylation rate increased linearly with an increase in leaf P per unit area (mg P m '), but rates were lower at elevated CO2 for a given P content Mi2. Vcmax also increased linearly with an increase in leaf P concentration (mg P gI structural dry weight). However, values of Vcmax were similar for plants grown at ambient and elevated CO2, for a given P concentration. Acclimation of photosynthesis at elevated C02 was associated with an increase in leaf starch determined 5 h into the light period. However, increased starch concentration with an increase in P supply was not associated with any decline in Vcmax (Barrett and Gifford, 1995).




16
Effect of Water Stress on Photosynthesis
One of the major injurious responses to water deficits is nonstomatal inhibition of photosynthesis, that is, inhibition of photosynthesis that cannot simply be ascribed to stomatal closure (Hsiao, 1973).
Inhibition of leaf photosynthesis may result from a decrease in the conductance of CO2 from the atmosphere to the chloroplasts, such as occurs when stomata close; from detrimental effect on the photosynthetic mechanism (mesophyll activity) itself; or from a combination of the two (Schulze, 1986). Leaf rolling is a common response by grasses to a water stress. This reduces water used, but also inhibits C02 assimilation per plant (Schulze, 1986).
Boyer (1976) indicated that plant sensitivity to drought varies with the stage of growth. When leaves begin to desiccate, photosynthesis is inhibited and can be affected enough so that net carbon dioxide fixation ceases completely. Moreover, Yoshida (1978) reported that water stress affects both stomatal and nonstomatal components of photosynthesis of rice. Initial photosynthetic reduction is due to a decrease in the conductance of C02 through stomata arising from plant water deficit. Furthermore, water stress reduces net photosynthate availability by reducing leaf area. This is followed by a decrease in the activities of enzymes such as RuBPCase and in the photochemical activity of the chloroplast (Sinha et al., 1982) Several investigators have demonstrated




17
changes in chloroplast activity when chloroplasts are isolated from desiccated leaves. These changes involve a decrease in electron transport and photophosphorylation, and there are reports that CO2 fixation by isolated chloroplasts is reduced (Boyer, 1976).
At minimal least water potential, sorghum (Sorghum bicolor L.) net photosynthesis was completely inhibited, with the stomata being closed (Contour-Ansel et al., 1996). After rewatering, plants showed recovery in photosynthesis but never reached the initial values. Water stress had a striking effect, both on net photosynthesis by regulation of stomatal aperture and Pyruvate P, Dikinase (PPDK) and Phosphoenol Pyruvate Carboxylase (PEPC) activities. Therefore, the level of PPDK and PEPC activities may contribute to the limitation of photosynthetic CO2 fixation.
Clifford et al. (1995) found that CO2 exerted significant effects on groundnut stomatal frequency only in irrigated groundnut plants. The effects of drought on leaf development outweighed the smaller effects of [CO2], although reductions in stomatal frequency induced by elevated atmospheric C02 were still observed. Elevated atmospheric CO2 promoted larger reductions in leaf conductance than the changes in stomatal frequency, indicating partial stomatal closure. As a result, the plant stands grown at elevated CO2 utilized the available soil moisture more slowly than those grown under ambient CO2, thereby possibly extending the growing period.




18
Samarakoon and Gifford (1996a), studying cotton (Gossypium hirsutum cv. Sicala 34), found high-CO2 plants decreased transpiration rate by 60% compared to that of lowCO2 plants under water deficit. In addition, in wet soil maize transpiration rate was reduced on average by 29% at high C02, but neither total dry matter nor plant height were significant .y affected by CO2 level (Samarakoon and Gifford, 1996b). In 3oil that was drying from field capacity, plants in high CO2 used about 30% less water than those in ambient CO2.
Kameli and Losel (1996) reported that inhibition of growth of wheat (Triticum c ,rum L.) was only apparent when the water content of the plant started to decline. Dry weight of wheat continued to increas during water stress. This caused a sharp rise in sugar cont, it, accounting for 20% of the gain in dry matter between days 27 and 31. Following re-watering, stressed plants increased leaf length and leaf area, and leaves regained turgidity after wilting. Growth inhibition coincided with a considerable increase in sugar content. Photosynthesis rather than reserve starch was proposed to be the major source of sugar accumulated under water stress in durum wheat.
Tomlinson and Anderson (1995) reported that seedling biomass of red oak (Quercus rubra L.) increased with
increasing CO2 and decreased with water stress. Water stress shifted relative biomass distribution from stems to roots,




19
whereas CO2 did not alter distribution among leaves, stem, or roots. Photosynthetic rate increased with increasing Co2 and decreased with water stress. Stomatal conductance decreased with both water stress and elevated CO2. Photosynthetic wateruse efficiency was greater at elevated growth CO2 but largely unaffected by water stress.
Delgado et al. (1992) observed different patterns of photosynthesis in stressed and non-stressed plants of Nicotiana tabacum L. for which water stress reduced total net carbon fixation by 45%. Moreover, the decrease in dry mass production under water stress was related to a decrease in total leaf area per plant and decrease in cell number per unit leaf area.
Jones (1993) reported that under irrigated conditions net photosynthesis of groundnut (Arachis hypogaea) leaves increased by approximately 40% when C02 was increased from 350 to 700 ppmv. Droughted plots in both CO2 regimes exhibited negligible net photosynthesis after 85 DAS. In addition, Clifford et al. (1993) observed that with groundnut grown in well-irrigated conditions, elevated CO2 increased dry matter accumulation 15% and pod yields 30% (from 2.8 to 3.7 t ha 1) Under drought condition, elevated CO2 increased groundnut dry matter production by 112% and yield by 468% (from 0.22 to 1.25 t ha 1) as compared to plants grown at 350 ppmv C02.




20
Effects of CO and Water Stress on
Non-Structural Carbohydrates
Growth under long-term C02 enhancement can lead to carbohydrate accumulation (Allen et al., 1988; Baker et al., 1992; Rowland-Bamford et al., 1990; Wong, 1990). This may be because the photosynthetic rate exceeds the sink capacity to utilize the photosynthate for growth. An apparent correlation between starch accumulation and suppression of photosynthesis has been often reported (Sasek et al., 1985). Although extreme enlargement of starch grains may lead to physical damage of the chloroplast (DeLucia et al., 1985) and also hinder CO2 diffusion in the chloroplast, there is no evidence that starch accumulation directly inhibits photosynthesis. Another explanation is that starch accumulation in the chloroplast may occur when photosynthesis is suppressed by decreased capacity of orthophosphate (Pi) regeneration during starch and sucrose synthesis.
Water-stressed leaves have decreased rates of starch synthesis and increased synthesis of sugars (Morgan, 1984). The author sugested that it is possible that changes in enzyme activities of the pathway leading to starch synthesis play a substantial role in altering partitioning during stress.
When attached, translocating bean leaves were water stressed and partitioning was assayed by 10-min pulselabelling with 4C02, starch accumulation was decreased by more than 75% (Vassey and Sharkey, 1989). In contrast, the




21
accumulation of 4C into the neutral fraction, which includes sucrose, was not significantly affected. Only a small
fraction of [4C]sucrose would be exported via phloem during the 10-min pulse duration in this study. Vassey and Sharkey (1989) showed that the ionic fraction, which includes organic acids and amino acids, increased from 4% (control) to 42% (stressed) of the recovered "4C. Rhodes (1987) concluded that stressed leaves are known to synthesize proline and glycine betaine.
Sucrose synthesis
Sucrose synthesis is inhibited when sucrose accumulates in the leaf (Foyer, 1990), and this feedback inhibition seems to stimulate starch synthesis. Since feedback inhibition of sucrose synthesis leads to P1-regeneration limited photosynthesis in the short-term regulation, it is possible that starch accumulation may be an effective indicator of Piregeneration limited photosynthesis. However, starch synthesis in the chloroplast also frees Pi, and actually starch accumulation can occur without suppression of photosynthesis (Stitt, 1991).
In the flag leaf of wheat (Triticum aestivum) (Nie et al., 1995), soluble carbohydrate concentrations declined markedly with the onset of grain filling. However, carbohydrates that were stored in vegetative plant parts before heading made a smaller contribution to grain dry weight




22
at [CO2] below 330 jimol mol than for treatments at above
ambient [C02], and increasing [CO2] had no effect on the carbohydrate concentration in the grain at maturity (RowlandBamford et al., 1990).
Plants of Scirpus olneyi grown at elevated [CO2]
contained more non-structural carbohydrates (25-53%) than those grown at ambient [CO2] (Jacob et al., 1995). Plants grown at elevated [CO2] appear to have sufficient sink capacity to utilize the additional carbohydrates formed during photosynthesis. Wang and Nobel (1995) reported that doubling the [C02] led to approx. 5% more sucrose, 560% more mannose and 17% less amino acids in the phloem exudate and also significantly increased mannose, starch and glucomannan in the chlorenchyma of Opuntia ficus-indica. Moreover, Sicher et al. (1995) revealed that leaf starch and sucrose levels were greater in soybean plants grown at 70 than at 35 Pa CO2.
Partitioning into Starch and Sucrose
Sucrose and starch are the principal end products of photosynthesis in most plants, and sucrose is the principal carbohydrate translocated from source to sink tissues (Stitt et al., 1987). During the day leaves accumulate sucrose and starch as well as exporting sucrose to the rest of the plant. At night the sucrose and starch stored during the day are mobilized to maintain export of sucrose to sink tissues and to support respiration in the leaf (Servaites et al. 1989). The




23
types of carbohydrate stored in the leaves varies between species. In many species (e.g. soybean, potato and tomato) starch is the major leaf storage carbohydrate, while other species store either sucrose and starch (e.g. spinach) or mainly sucrose (e.g. wheat and barley) (Stitt et al., 1987).
Lunn and Hatch (1995) found that ratio of primary partitioning into sucrose and starch varied from about 0.5 in those species that accumulated mostly starch in the leaves (Amaranthus edulis L., Atriplex spongiosa F. Muell. and Flaveria trinervia (Spreng.) C. Mohr) to about 8 in Eleusine indica (L.) Gaertn., which accumulated mostly sucrose.
Generally there was a reasonable link between the primary partitioning of photosynthate and the type of carbohydrate stored in the leaf during the day.
It is also generally recognized that the starch content of Trifolium subteraneum leaves increases with increasing CO2 concentration (Cave et al., 1981), while Zhang and Nobel (1996) reported that sucrose and starch contents of Encelia farinosa, a common C3 desert shrub, increased during the day proportionally more than under the ambient CO2.
Galtier et al. (1995) reported that the rate of sucrose synthesis was increased relative to that of starch and sucrose/starch ratios were higher throughout the photoperiod in the leaves of all tomato plants expressing high SPS activity.




24
Neutral sugars (predominantly sucrose) of wheat (Triticum aestivum) were found to be the most abundant form of carbohydrate accumulated by leaves during the day, but significant quantities of starch and high degree of polymerization (d.p.) fructans were also present. Elevated CO2 was found to have marked effects on diel patterns of export, storage and respiration, while the proportion of fixed carbon allocated to each of these processes were broadly similar (Balaguer et al., 1995).
Combining high CO2 and different levels of nitrogen (N) and phosphorus nutrition treatments on pea (Pisum sativum), Riviere-Rolland et al. (1996) found that phosphoenolpyruvate (PEP) carboxylase decreased, and chloroplast phosphate (P)translocator increased, in high CO.. In contrast to Rubisco, down-regulation of PEP carboxylase was alleviated by low N and enhanced by low P. The increase in the P-translocator was little affected by N but was accentuated by low P. The
increase in the P-translocator is considered to be one way of alleviating low P condition in the chloroplast and thus rebalancing carbon partitioning between starch and soluble carbohydrates and amino acids. Riviere-Rolland et al. (1996) proposed that acclimation of PEP carboxylase and Ptranslocator reflects adaptation to metabolic perturbations caused by high C02.
There are conflicting reports as to the extent to which the extra carbon fixed (as a result of CO2 enrichment) is used




25
for export compared to storage (as starch) in leaves. Ho (1977) reported that enriched plants have a higher efficiency of carbon transport when transferred to, or grown at, elevated CO2. In contrast, Finn and Brun (1982) suggested that the majority of the additional reduced carbon provided by CO2 enrichment of soybean plants, was stored as leaf starch and was not available for transport to distant sink (roots and nodules). Starch and sucrose are the principal end-products of photosynthesis (Silvius et al., 1979 and Preiss, 1982). It has been postulated that the rate of sucrose formation indirectly controls the rate of starch formation (Silvius et al., 1979). Starch is deposited during the day exclusively in the chloroplast (Preiss, 1982).
Patel and Mohapatra (1996) reported that sucrose was the major translocatable sugar in the organs of fertile rice florets of both top and basal spikelets, and poor grain filling of the latter was not caused by deficiency of sucrose, but due to inhibition of sucrose supply of the external protective organs.
Based on 4CO2 pulse-chase experiment on Phaseolus vulgaris, Sharkey et al. (1985) reported that sucrose formation was linearly related to assimilation rate (slope=0.35), while starch formation increased linearly with assimilation rate (slope=0.56) but did not occur if the assimilation rate was below 4 1xmol m2 s". They indicated that the pathways for starch and sucrose synthesis are both




26
controlled by the rate of net C02 assimilation, with sucrose the preferred product at very low assimilation rates.
Nie et al. (1995) found that differences in soluble carbohydrate concentration between wheat leaves grown at
elevated and current ambient C02 concentration diminished with crop development, while in rice leaf blades, the priority between the partitioning of carbon into storage carbohydrates or into export changed with developmental stage and [Ca2] (Rowland -Bamford et al., 1990). Moreover, during vegetative growth of rice, leaf sucrose and *starch concentrations increased with increasing [C021 but tended to level off above 500 limol1 mol1 CO2. The ratio of starch t o sucrose concentration was positively correlated with the ICa2]. At maturity, increasing [CO2] resulted in an increase in total non-structural carbohydrate concentration in leaf blades, leaf sheaths and culms of rice (Rowland-Bamford et al., 1990).
In C3 plants, elevated atmospheric CO2 concentrations can partially compensate for the negative effects of drought by increasing water-use efficiency and by sustaining larger net CO2 assimilation rates at reduced stomatal conductance in leaves of stressed plants (Chaves and Pereira, 1992) However, studies of rice in response to both elevated [CO2] and drought stress during the reproductive phase have not been conducted.
Therefore, this study contains three objectives to investigate the effects of enhanced [C02] and water regimes on




27
photosynthesis and photosynthate partitioning of rice leaves at panicle initiation and/or anthesis. Objective 1. To examine how leaf photosynthesis, total soluble
protein and total chlorophyll in leaves is affected by
water stress and elevated C02.
Objective 2. To study effects of elevated-C02 and water
stress on total non-structural carbohydrates in leaves. Objective 3. To determine sucrose phosphate synthase activity
in leaves affected by water stress and C02-enrichment.




CHAPTER 2
EFFECTS OF ELEVATED [C02] AND WATER DEFICIT ON RICE LEAF PHOTOSYNTHESIS, CHLOROPHYLL CONTENT AND LEAF SOLUBLE PROTEIN Introduction
Atmospheric CO2 enrichment generally enhances photosynthesis and plant growth rates (Kimball, 1983). Baker and Allen (1993) found that the direct effect of [C02] enrichment on rice (Oryza sativa L.), soybean (Glycine max L.) and citrus (various species) was always an increase in photosynthetic rate. Furthermore, it was reported that photosynthetic rate of white clover (Trifolium repens L.) (Ryle et al. 1992a) and perennial ryegrass (Lolium perenne L. cv. Melle) (Ryle et al. 1992b) increased by 17-29% and 35-46%, respectively, with elevated (680 ppmv) [C02] treatment compared to 340 ppmv [CO2] Arp and Drake (1991), studying Scirpus olneyi grown in elevated C02, found an increase in photosynthetic capacity by 31%. Short-term and long-term CO2 enrichment of soybean plants resulted in increased rates of leaf photosynthesis (Huber et al., 1984; Valle et al., 1985; Campbell et al., 1990) and canopy photosynthesis (Jones et al., 1984a). Three seed crops, two forage crops and two native plant ecosystems grown in elevated CO2 demonstrated increased canopy photosynthesis (Drake and Leadley, 1991).
28




29
Baker et al. (1990b) and Rowland-Bamford et al. (1991) reported that rice photosynthetic rates increased with increasing [CO] treatment from 160 to 500 ppmv followed by a leveling off of the response among the superambient [C02] treatments. Rice photosynthetic and growth response to elevated CO2 is reported to be highly dependent on the supply of N. Moreover, if additional CO2 is given and N is not available, lack of sinks for excess carbon (e.g. tillers) may limit the photosynthetic and growth response (Ziska et al., 1996).
Total soluble protein and leaf chlorophyll content of three-week-old soybean were reported to be unaffected by 70 Pa and 35 Pa CO2 treatments (Sicher et al., 1995). Radoglou and Jarvis (1992) as well as Graham and Nobel (1996) reported that chlorophyll content per unit leaf area was less under elevated [C02] for Phaseolus vulgaris and the CAM plant, Agave deserti, respectively. Rowland-Bamford et al. (1991) reported that rice leaf nitrogen content decreased and rubisco protein as a fraction of total soluble leaf protein decreased by 32% with elevated C02.
Water stress decreases net photosynthetic rate in crop plants and this decrease has previously been attributed largely to a decrease in stomatal conductance which restricts exogenous C02 supply and thereby changes the balance between carboxylation and oxygenation by Rubisco (Boyer and McPherson, 1981; Farquhar and Sharkey, 1982). In contrast, Di Marco et




30
al. (1988) studying wheat (Triticum durum L.) concluded that C02 supply was not limiting, because the ratio of intercellular [C02] (C,) to ambient [CO] (Ca) for the stressed plants was similar to the irrigated control. Also, the
maximal rate of photosynthesis in saturating CO2 of stressed plants was quite similar to the rate of photosynthesis under natural conditions. Allen et al. (1994) also showed that the Ct/Ca ratio of soybean leaves did not change throughout a drought cycle. Although C02 was not limiting, Irigoyen et al. (1992) reported leaf water potential lower than -2.8 MPa directly affected CO2 fixation.
Water-stressed plants have lower leaf water potential and stomatal conductance. Further stress causes leaves to desiccate and therefore photosynthesis becomes inhibited and can be affected enough so that net carbon dioxide fixation ceases completely (Boyer and McPherson, 1981).
Pea (Pisum sativum L. cv. Frilene) plants subjected to drought (Total leaf water potential=-l.3 MPa) were reported to have major reductions in photosynthesis (78%) and minor reduction (=18%) in the contents of chlorophyll a, carotenoid, and soluble protein (Moran et al., 1994). Furthermore, two cultivars of bean (Phaseolus vulgaris L.) decreased in soluble protein and chlorophyll content after drought stress was applied. In that study, chlorophyll content increased above control value and protein content also increased during the re-watering period (Castrillo and Trujillo, 1994).




31
In drying soil, maize (Zea mays L.) photosynthesis became responsive to high C02, thus resulting in a considerable increase in dry matter and leaf area (Samarakoon and Gifford, 1996b). Gifford (1979) studying drought stressed wheat, found that elevated [C02] increased dry matter production due to increased water-use efficiency and resulted in adaptation to water stress through osmoregulation.
Chaudhuri et al. (1986) reported that winter wheat under either drought stress or full irrigation and high [C02] has higher chlorophyll content compared to those of low [CO2].
Little is known about rice responses to the combination of CO2 enrichment and water deficit during the reproductive period, but it is a C3 species whose photosynthesis is responsive to increases in [CO2] (Baker et al., 1996a, 1996b; Ziska et al., 1996), and rice, however, is very sensitive to water stress during the reproductive phase (Yoshida, 1978; Baker et al., 1996a, 1996b). The objectives of this study were to determine the responses of rice photosynthesis under various water management treatments in long-term doubling of [C021.
Materials and Methods
Plant material. The research was conducted during the 1994 growing season (15 July to 24 November) at the Irrigation Research and Education Park of the University of Florida at Gainesville, Florida (Baker et al., 1996a, 1996b). Rice




32
(Oryza sativa L. cv. IR-72) plants were grown in eight sunlit, controlled environment chambers. The chambers consisted of clear "sixlight" tops and walls 2.0 by 1.0 m in cross section by 1.5 m in height, attached to 0.6 m deep heavy-gauge bins filled with soil to a depth of 0.5 m. The soil used was fine sand (loamy, siliceous, hyperthermic Grossarenic Paleudult). A detailed description of the growth chamber design and computer control system may be found in Jones et al.(1984a, 1984b) while Baker et al. (1990a, 1990b) provide information on the modifications for growing rice. Pickering et al. (1993) described the current chamber system. The chambers were exposed to natural sunlight. Four chambers were controlled at 350 pL C02 L-' and 4 chamber were controlled at 700 pL CO2 L'. In each pair of chambers (one ambient and one elevated [C021), the following water management-stress regimes were imposed: a) continuously flooded (FLD), b) paddy flood water removed (soil bins drained) and drought stress imposed during panicle initiation (DPI), c) drought stress imposed during anthesis (DAN), and d) drought stress imposed at both panicle initiation and anthesis (DBS) (Table 2.1). Dry bulb air temperature and dew point were controlled to 28/210 (day/night) and to 18/12 C (day/night), respectively.
Seeds of rice (cv. IR-72) were sown at rate of 200/m2 in 11 rows 18 cm apart on 15 July 1994. After the seeds germinated and rice plants had one leaf, flood water was added daily in increments of 1 cm above the soil surface until water




33
achieved a height of 5 cm where it was maintained. When rice
plants reached growth stages for initiating drought treatments listed above, then paddy water was drained. When rice leaves
started curling and desicating to the point of zero canopy assimilation, water was restored as shown in Table 2.1. Further information on whole-canopy photosynthesis and
evaporation during the imposed droughts, and growth and yield are given by Baker et al. (1996a, 1996b). Because the soil bins provided an absolute boundary and rice roots permeated
the entire volume, expression of severe drought came suddenly and swiftly to the plants as the soil bins ran out of plantavailable soil water. It is important to note that the drought stress cycles were initiated by draining paddy water from the bottom of the soil bins. At the early stages of the
imposed drought cycle, the soil was essentially in equilibrium with a very shallow water table (about 1-cm deep) at the bottom of the soil bins. Tensiometers in the soil did not indicate large soil water tensions until near the end of the drought cycle (Baker et al., 1996a, 1996b).
Photosynthetic measurements. Photosynthetic measurement was conducted on three fully-expanded leaves from each chamber begining at 12:00 noon eastern standard time. Measurements were taken on various dates before drought, during drought and after re-watering. The LICOR LI-6250 Portable Photosynthesis System with 0.25-L chamber were used to measure
photosynthetic rate at the corresponding CO2 concentrations.




34
Each data was computed from 3 measurements which each
measurement consisted of 2 data.
Table 2.1. Times when water was withheld and restored for
drought treatments for all chambers. Rice was planted on
15 July 1994.
CO2 WM Stress at P.I. Stress at Anthesis
WW WR WW WR
pL L- DAP DAP----350 FLD f t t t
DPI 57 74 t t
DAN t t 93 110
DBS 57 74 96 ill
700 FLD t t t t
DPI 57 75 t t
DAN t t 93 110
DBS 57 75 96 i1
Note:
FLD: continuously flooded DPI: drought imposed during panicle initiation DAN: drought imposed during anthesis DBS: drought imposed during both stages DAP: days after planting WM : water management
WW : water withdrawn
WR : water restored
t: paddy water still present PI : Panicle Initiation
Total soluble protein. For soluble protein and
chlorophyll concentration analysis, 15 fully-expanded leaves
were excised and rapidly immersed in liquid N2 around 12:00
noon eastern standard time on various dates before drought,
during drought and after re-watering. These same leaf samples
were used for sucrose phosphate synthase analysis in Chapter
4. Leaves were collected from both the eastern half and
western half of each chamber and combined.




35
Leaf samples were preserved in liquid nitrogen until analysis. For soluble protein and chlorophyll assay, 0.1 g of frozen leaf was weighed and ground in chilled mortar using a 1:5 tissue- to-buf fer ratio in a medium containing 5 ml of MOPS buffer at pH 7.5. After centrifugation, total soluble protein
in an aliquot of the extract was determined wi th the dye
binding method (Bradford, 1976) using gamma-globulin as the
standard. For soluble protein and chlorophyll, each data was calculated from two grinds out of one pooled sample. Each grind was divided for two extractions and each extraction was used for four assays.
Leaf chlorophyll content. The leaf extract obtained
from protein analysis above was used to measure chlorophyll concentration. Chlorophyll was extracted by diluting the extract in 80% acetone for 15 min in the dark at 40C. After centrifugation, the chlorophyll content was determined using
the method of Yoshida et al. (1972), measuring the absorbance at 645, 652 and 663 rim using a spectrophotometer.
Results and Discussion
Photosynthetic rate (Pn). The photosynthetic rate
(Pn) of leaves of rice grown at 350 and 700 1.1 CO2 1"' and in various water management treatments are given in Tables 2.2 through 2.5. The C02-enriched plants exhibited higher Pn at each sampling date throughout the season. Before drought was imposed, the treatment differences were only attributed to CO2




36
level. Under continuously-flooding, the doubled-CO2
treatments increased leaf photosynthetic rate by 40% from panicle initiation right through to final measurement before harvest (127 DAP).
The results obtained in this study indicate that CO2 enrichment increased photosynthetic rate (Pn) throughout the season in rice leaves when measured under continuously flooded water management. Increase of 50% in photosynthetic rate as a result of elevated CO2 has been reported for rice plants, exposed to 660 compared to 330 libar (Ziska and Teramura, 1992b; Baker et al., 1990b). Balaguer et al. (1995) and Jacob et al. (1995) also observed increases of 47 and 45% in Pn as a result of doubled-C02 for Triticum aestivum and Scirpus olneyi, respectively. The increases in Pn between the elevated and ambient CO2 treatment were slightly less
pronounced at the end of the growing season.
On the date of peak water deficit during the panicle initiation phase, leaf photosynthetic rate was decreased by 53% in enriched-C02 and 96% in ambient-C02 plants (at 74 DAP), while water deficit during anthesis decreased Pn by 89% in enriched-CO2 and 97% in low-CO2 plants (at 114 DAP). A
decrease was calculated as the difference between continuously-flooded treatment and corresponding water deficit and [C021 treatments (Table 2.3 and 2.5).
At 72 DAP (Table 2.2), 11 days after water was removed, drought had already affected leaf photosynthetic rate (Pn) of




37
plants grown at low CO2, but drought had not affected Pn of the high CO2 treatment. At 74 DAP, plants grown at 700 pL CO2 L I still maintained moderate Pn (14.7 and 10.6 Umol CO2 m2 s 1) while leaves grown at ambient CO2 had very low Pn (0.9 and 3.8 V=ol CO2 m 2 s '). After re-watering at the end of day 74 (Table 2.3), plants grown at low [C021 required a longer time to recuperate from drought stress. At 84 DAP (Table 2.3), the 700DPI treatment had reached the Pn rate of 700FLD, but the Pn rate of the 350DPI treatment was still below the Pn rate of 350FLD. The lingering effect of drought on Pn rate seemed to continue through 89 DAP and perhaps 98 DAP (Table 2.4).
At 109 DAP (Table 2.5), 16 days after water was withheld in 350DAN and 700DAN, photosynthetic rate of stressed leaves in ambient CO2 was significantly lower than the rate of leaves of stressed plants in elevated CO2. At the same time, 350DBS and 700DBS still maintained higher Pn compared to the two previous treatments. This may be due to 3-day delay in withholding water in 350DBS and 700DBS treatments. By 114 DAP (Table 2.5), 4 days after water was restored in 350DAN and 700DAN treatments and 3 days after water was restored in 35ODBS and 700DBS treatments, Pn of the later treatments was significantly higher than the rate of former treatments.
Ability of plants grown in high CO2 to maintain high Pn after imposing water deficit was due to the effects of high CO2 on water use efficiency (WUE). A number of studies have shown that elevated C02 reduces transpiration, primarily




38
through a decrease in stomatal conductance (Valle et al., 1985; Rogers et al., 1992). Baker et al. (1990b) reported a decrease in evapotranspiration and found increases in WUE with increasing [C02] in rice. Rogers et al. (1992) reported that water use efficiency (the ratio of C gain to water loss) increased substantially in elevated CO2 concentration.
Leaf soluble protein concentration. Leaf soluble protein from rice grown at 350 and 700 v.L CO2 L' and in various water management treatments is listed in Table 2.6 and 2.7. Leaf soluble protein of C02-enhanced plants were significantly lower compared to those grown at ambient CO2 treatment at 54, 82, 100, 110, and 127 DAP (P<0.05). The largest difference of leaf soluble protein concentration of plants grown in high-CO2 and ambient-CO2 chambers under continuously flooded treatment was by 18%.
Rowland-Bamford et al. (1991) observed a 32% decrease in leaf soluble protein of rice with CO2 enrichment and Jacob et al. (1995) reported a 45% decrease in soluble protein of Ficus opuntia. However, no effects of C02-enrichment on leaf soluble protein were reported on 3-week-old soybean plants grown in 35 Pa and 70 Pa CO2 (Sicher et al., 1995) and 58 dayold soybean plants grown in 330, 450 and 600 umol mol1 (Allen et al., 1988).
Table 2.6 and 2.7 show that water stress effects on leaf soluble protein concentration were significant at 74, 82, 92, 110 and 127 DAP. After paddy water was removed from 350DPI,




39
350DBS, 700DPI and 700DBS treatments at 57 DAP to initiate stress treatment at panicle initiation, a decrease in leaf soluble protein concentration was detected at 74 DAP (Table 2.6) in elevated-C02 plants and ambient-C02 plants. The
decrease was 62 and 56% in 350DPI and 700DPI treatments, respectively.
water was restored at 74 DAP in 350DPI and 350DBS treatments and at 75 DAP in 700DPI and 700DBS treatments. By 92 DAP the protein concentration of the stressed plants had reached the level of leaf soluble protein concentration of continuously-flooded treatments (Table 2.6).
For stress treatment at anthesis, paddy water was drained from chambers at 93 DAP for treatments of 350DAN and 700DAN, and at 96 DAP for treatments 350DBS and 700DBS. By 100 DAP (Table 2.7), leaf soluble protein concentration was not yet affected by water stress treatment (P=0.5474). By 110 DAP the water management treatments were significantly different (P<0.01) and a decrease in leaf soluble protein concentration was detected (Table 2.7). The decrease in leaf soluble protein content was 62 and 55% in 350DAN and 700DAN, and 56 and 53% in 350DBS and 700DBS, respectively.
Leaf Chlorophyll Concentration. Leaf chlorophyll
concentration of rice leaves grown at 350 and 700 UL C02 L" and for various water management treatments are given in Table 2.8 and 2.9. Chlorophyll concentration was significantly different with (Co.) treatment from 54 to 110 DAP (P<0.05).




40
In addition, the water management treatments were significantly different at 74 and 110 DAP (P<0.05).
Average increase chlorophyll concentration for enrichedC02 over ambient-CO2 plants was detected under continuouslyflooded water treatment by 13%, but there was no increase at 110 nor 127 DAP (Table 2.9). Decreases in chlorophyll
concentration of 62 and 73% in ambient-C02, and 48 and 54% in elevated-CO2 leaves were observed during the water stress period at panicle initiation (74 DAP) and anthesis (110 DAP), respectively. Repeated water stress effects were significant and decreased chlorophyll concentration by 70% in low-CO2 and 55% in doubled-Co2 plants.
Increase in leaf chlorophyll content of plants grown in elevated-CO has also been found in soybean leaf blades (Allen at al., 1988). In addition, increase in chloroplast density or volume has also been reported to occur in soybean plants grown in an elevated Co2 environment (Thomas and Harvey, 1983; Vu et al., 1989). By contrast, decrease of chlorophyll content of elevated C02 plants has been observed on pea (Xu et al., 1994), soybean (Radaglou and Jarvis, 1992), tomatoes (Khavari-Nejad, 1986) and other species (Delucia et al., 1985).
Water deficit caused major reduction of chlorophyll concentration at panicle initiation at 74 DAP (Table 2.8). Decreases in chlorophyll were 62% and 48% in 350DPI and 700DPI plants, respectively. Reductions due to water stress at




41
anthesis, 110 DAP (Table 2.8), were 73 and 76% in 350DAN and 350DBS, and 54 and 63% in 700DAN and 700DBS treatments, respectively.
The decrease in chlorophyll content after drought stress has been reported on wheat (Chaudhuri et al., 1986), however, after re-watering, the chlorophyll content increased above control value (Castrillo and Trujillo, 1994).
Based on the above results, doubled-[CO2] treatments in rice had significantly increased leaf Pn and chlorophyll concentration, but decreased leaf soluble protein concentration. Withholding water during either panicle initiation or anthesis decreased Pn, leaf soluble protein and chlorophyll concentration.




42
Table 2.2. Leaf photosynthetic rate of rice plants grown at 350 and 700 IiL CO2 LI and in four different water managements. Measurements were taken on 51, 55, and 60 day-old plants (before stress affected) and 72 day-old plants (early water deficit period). Photosynthesis was measured with the LICOR LI-6250 using 0.25-L chamber with corresponding CO2 concentrations.
C02 Water Leaf Photosynthetic Rate
Management 51DAP 55DAP 60DAP 72DAP
UL L ---nl CO2 m2 s
350 FLD 23.83.0 24.31.2 24.30.7 25.42.1
DPI 24.00.4 27.31.5 27.12.1 24.72.6
DAN 23.93.1 25.31.4 26.02.6 24.72.5
DBS 22.21.9 26.81.2 23.41.2 18.81.3
700 FLD 30.41.9 33.41.0 34.82.2 36.52.5
DPI 30.51.5 36.02.1 32.71.9 29.50.3
DAN 32.24.5 32.22.0 28.62.2 41.22.3
DBS 30.33.9 32.11.4 26.41.7 24.61.8
CO2 P=0.0001 P=0.0001 P=0.0024 P=0.0001
WM P=0.1417 P=0.0740 P=0.1121 P=0.0001
C02*WM P=0.6430 P=0.3962 P=0.2713 P=0.0264
Note:
FLD: continuously flooded DPI: drought imposed during panicle initiation DAN: drought imposed during anthesis DBS: drought imposed during both stages DAP: days after planting WM : water management
Paddy water was removed at 57 DAP to lead to water stress at panicle initiation in 350DPI, 350DBS, 700DPI and 700DBS treatments.
Water was restored after sampling at 74 DAP in 350DPI and 350DBS treatments, and at 75 DAP in 700DPI and 700DBS treatments.




43
Table 2.3. Leaf photosynthetic rate of rice plants grown at 350 and 700 UL CO2 LI and in four different water managements. Measurements were taken at noon eastern standard time from 74 (stressed), 75 (stressed), 82 and 84 (recovery) day-old plants. Photosynthesis was measured with the LICOR LI-6250 using 0.25L chamber with corresponding C02 concentrations. On 75 DAP, treatment 350DPI and 350DBS had been re-watered but 700DPI and 700DBS had not yet been watered.
CO2 Water Leaf Photosynthetic Rate
Management 74DAP 75DAP 82DAP 84DAP
jL L-- Imol CO2 m2 Si
350 FLD 22.20.4 20.11.8 23.72.0 21.91.0
DPI 0.91.2 12.52.9 17.32.1 17.22.3
DAN 24.11.9 21.51.2 22.41.4 23.11.9
DBS 3.81.0 13.51.3 17.91.3 18.91.5
700 FLD 31.12.5 28.80.8 33.30.7 33.92.6
DPI 14.73.7 1.11.5 30.62.3 33.02.7
DAN 30.31.0 27.22.7 31.52.2 34.01.2
DBS 10.63.0 5.42.9 29.61.4 32.92.7
-------------------------------------------------CO2 P=0.0001 P=0.0015 P=0.0001 P=0.0001
WM P=0.0001 P=0.0001 P=0.0005 P=0.0818
CO2*WM P=0.0692 P=0.0001 P=0.0233 P=0.0958
Note:
FLD: continuously flooded DPI: drought imposed during panicle initiation DAN: drought imposed during anthesis DBS: drought imposed during both stages DAP: days after planting WM : water management
Water was restored after sampling at 74 DAP in 350DPI and 350DBS treatments, and at 75 DAP in 700DPI and 700DBS treatments.




44
Table 2.4. Leaf photosynthetic rate of rice plants grown at 350 and 700 11L CO2 LI and in four different water managements. Measurements were taken on 89, 98 and 100 day-old plants. Photosynthesis was measured with the LICOR-6250 using 0.25-L chamber with corresponding CO2 concentrations. C02 Water Leaf Photosynthetic Rate
Management 89DAP 98DAP 100DAP
IjL L- mol CO2 m2 S-1
350 FLD 24.22.7 22.61.8 25.81.8
DPI 18.00.8 20.41.5 27.12.4
DAN 26.51.0 24.60.1 24.20.4
DBS 18.31.2 16.10.7 23.20.5
700 FLD 35.40.8 33.01.8 33.41.3
DPI 36.90.6 36.52.8 35.11.9
DAN 34.80.4 37.60.7 31.12.3
DBS 35.81.2 33.22.5 30.52.4
CO2 P=0.0001 P=0.0001 P=0.0001
WM P=0.0003 P=0.0001 P=0.0031
C02*WM P=0.0001 P=0.0134 P=0.9599
Note:
FLD: continuously flooded DPI: drought imposed during panicle initiation DAN: drought imposed during anthesis DBS: drought imposed during both stages DAP: days after planting WM : water management
Paddy water was removed at 93 DAP in 350DAN and 700DAN treatments, and at 96 DAP in 350DBS and 700DBS treatments. Water was restored at 110 DAP after sampling in 350DAN and 700DAN treatments, and at 111 DAP in 350DBS and 700DBS treatments.




45
Table 2.5. Leaf photosynthetic rate of rice plants grown at 350 and 700 IjL C02 L' and in four different water managements. Measurements were taken from 109, 114 and 125 day-old plants. Photosynthesis was measured with LICOR-6250 using 0.25-L chamber with corresponding CO2 concentrations.
CO2 Water Leaf Photosynthetic Rate
Management 109DAP 114DAP 125DAP
iL L -IMol CO2 m2 s 1
350 FLD 27.21.8 29.60.4 24.31.3
DPI 30.01.6 28.71.9 21.24.2
DAN 3.01.5 1.00.9 19.21.2
DBS 10.51.2 20.72.2 23.21.7
700 FLD 40.81.6 37.50.1 31.63.5
DPI 41.40.9 39.50.6 35.72.4
DAN 13.11.7 4.02.9 33.61.1
DBS 19.61.3 34.31.5 27.32.3
CO2 P=0.0001 P=0.0001 P=0.0001
WM P=0.0001 P=0.0001 P=0.2603
C02*WM P=0.0223 P=0.0002 P=0.0188
Note:
FLD: continuously flooded DPI: drought imposed during panicle initiation DAN: drought imposed during anthesis DBS: drought imposed during both stages DAP: days after planting WM : water management
Paddy water was removed at 93 DAP in 350DAN and 700DAN treatments, and at 96 DAP in 350DBS and 700DBS treatments. Water was restored at 110 DAP after sampling in 350DAN and 700DAN treatments, and at 111 DAP in 350DBS and 700DBS treatments.




46
Table 2.6. Leaf soluble protein concentration of rice plants grown at 350 and 700 pL CO2 L' and in four different water managements. Measurements were taken from 54, 74, 82 and 92 day-old plants.
CO2 Water Leaf Soluble Protein Concentration
Management 54DAP 74DAP 82DAP 92DAP
L L' ---- mg g' fresh weight
350 FLD 37.21.7 31.93.1 30.43.5 31.21.7
DPI 43.04.8 12.21.2 28.91.9 30.11.2
DAN 37.53.3 34.50.6 32.42.6 32.91.9
DBS 35.73.1 12.72.1 29.30.9 30.82.2
700 FLD 30.41.3 30.60.5 30.81.6 30.31.0
DPI 30.81.4 13.40.7 24.40.8 28.21.7
DAN 33.12.5 30.31.6 32.22.0 32.52.3
DBS 30.02.5 15.61.2 26.10.7 29.70.9
--------------------------------....---------------CO2 P=0.0001 P=0.6115 P=0.0320 P=0.0865
WM P=0.1119 P=0.0001 P=0.0005 P=0.0116
CO2*WM P=0.1188 P=0.0100 P=0.1369 P=0.9277
Note:
FLD: continuously flooded DPI: drought imposed during panicle initiation DAN: drought imposed during anthesis DBS: drought imposed during both stages DAP: days after planting WM : water management
Paddy water was removed at 57 DAP to lead to water stress at panicle initiation in 350DPI, 350DBS, 700DPI and 700DBS treatments.
Water was restored after sampling at 74 DAP in 350DPI and 350DBS treatments, and at 75 DAP in 700DPI and 700DBS treatments.




47
Table 2.7. Leaf soluble protein concentration of rice plants grown at 350 and 700 IiL CO2 L and in four different water managements. Measurements were taken from 100, 110 and 127 day-old plants.
CO2 Water Leaf Soluble Protein Concentration
Management 100DAP 1IODAP 127DAP
IiL L' ----- mg g fresh weight ----350 FLD 31.50.3 32.81.2 25.61.4
DPI 34.92.1 32.51.0 24.50.9
DAN 31.92.0 12.50.6 20.91.1
DBS 33.92.9 14.50.7 20.50.5
700 FLD 29.71.5 30.91.0 23.40.4
DPI 28.12.1 29.30.7 25.01.4
DAN 28.31.2 13.90.3 20.91.1
DBS 28.21.8 14.60.6 18.50.7
CO2 P=0.0001 P=0.0021 P=0.0445
WM P=0.5474 P=0.0001 P=0.0001
CO2*WM P=0.1714 P=0.1081 P=0.0703
Note:
FLD: continuously flooded DPI: drought imposed during panicle initiation DAN: drought imposed during anthesis DBS: drought imposed during both stages DAP: days after planting WM : water management
Paddy water was removed at 93 DAP in 350DAN and 700DAN treatments, and at 96 DAP in 350DBS and 700DBS treatments. Water was restored after sampling at 110 DAP in 350DAN and 700DAN treatments, and at 111 DAP in 350DBS and 700DBS treatments.




48
Table 2.8. Leaf chlorophyll concentration of rice plants grown at 350 and 700 )lL CO2 L-' and in four different water managements. Leaf samples were taken from 54, 74, 82 and 92 day-old plants.
CO2 Water leaf Chlorophyll Concentration
Management 54DAP 74DAP 82DAP 92DAP
pL L-' ----- mg g' fresh weight
350 FLD 4.10.2 3.70.5 3.30.1 3.70.7
DPI 3.80.2 1.40.3 3.30.6 3.90.4
DAN 4.10.1 3.80.6 3.70.2 3.70.9
DBS 3.80.1 1.30.5 3.30.9 3.61.2
700 FLD 4.70.2 4.40.4 4.01.2 4.01.0
DPI 4.11.1 2.30.6 4.00.8 4.01.2
DAN 4.10.6 4.00.8 4.10.4 4.00.1
DBS 3.90.1 2.30.3 4.00.7 3.90.2
CO2 P=0.0005 P=0.0004 P=0.0001 P=0.0185
WM P=0.0001 P=0.0001 P=0.2837 P=0.3691
C02*WM P=0.0049 P=0.2514 P=0.4540 P=0.7771
Note:
FLD: continuously flooded DPI: drought imposed during panicle initiation DAN: drought imposed during anthesis DBS: drought imposed during both stages DAP: days after planting WM : water management
Paddy water was removed at 57 DAP to lead to water stress at panicle initiation in 350DPI, 350DBS, 700DPI and 700DBS treatments.
Water was restored after sampling at 74 DAP in 350DPI and 350DBS treatments, and at 75 DAP in 700DPI and 700DBS treatments.




49
Table 2.9. Leaf chlorophyll concentration of rice plants grown at 350 and 700 uL C02 LI and in four different water managements. Measurements were taken from 100, 110 and 127 day-old plants.
CO2 Water Leaf Chlorophyll Concentration
Management 1OODAP IODAP 127DAP
UL L" ----- mg g9 fresh weight-.....
350 FLD 3.70.3 4.10.5 2.80.3
DPI 3.80.9 3.80.2 2.90.3
DAN 3.60.5 1.10.6 2.70.2
DBS 3.70.6 1.00.8 2.80.5
700 FLD 4.10.3 4.10.6 2.80.2
DPI 4.00.2 4.00.1 2.50.2
DAN 4.00.1 1.90.3 2.81.2
DBS 4.00.8 1.50.6 2.90.4
CO2 P=0.0192 P=0.0178 P=0.7084
WM P=0.8363 P=0.0001 P=0.9105
C02*WM P=0.9521 P=0.2943 P=0.3086
Note:
FLD: continuously flooded DPI: drought imposed during panicle initiation DAN: drought imposed during anthesis DBS: drought imposed during both stages DAP: days after planting WM : water management
Paddy water was removed at 93 DAP in 350DAN and 700DAN treatments, and at 96 DAP in 350DBS and 700DBS treatments. Water was restored after sampling at 110 DAP in 350DAN and 700DAN treatments, and at 111 DAP in 350DBS and 700DBS treatments.




CHAPTER 3
RICE LEAF NON-STRUCTURAL CARBOHYDRATES IN RESPONSE TO
ENHANCED CO2 AND WATER STRESS
Introduction
Sucrose and starch are the principal end products of photosynthesis in most plants, and sucrose is the principal carbohydrate translocated from source to sink tissues. During the day, leaves accumulate sucrose and starch as well as export sucrose to the rest of the plant (Preiss, 1982; Stitt et al., 1987). Leaf starch is an insoluble polyglucan that is deposited during the day-light hours exclusively in the chloroplast (Preiss, 1982). Conversely, sucrose is a watersoluble disaccharide that is synthesized in the cytosol and is the main form of reduced carbon translocated from source leaves to developing growth centers of the plant (Avigad, 1982). It is well established that sucrose synthesis is decreased when sucrose accumulates in spinach leaves (Stitt et al., 1987); however, Foyer (1987) suggested that the reduction in the capacity for sucrose synthesis caused by the buildup of sucrose is still questionable.
Accumulation of starch in the leaves is a commonly reported response for plants grown in elevated C02 (Arp, 1991). Plants such as soybean (Glycine max) (Havelka et al.,
50




51
1984; Allen et al., 1988), tomato (Lycopersicon esculentum) (Yelle et al. 1989), bean (Phaseolus vulgaris) (Hoddinot and Jolliffe, 1988) and rice (Rowland-Bamford et al., 1990) showed an increase in leaf starch content after long-term (days to months) treatment with elevated CO2. Rice leaf starch
concentration tended to level off above 500 Umol molI CO2. Short-term enhancement of [C021 was reported to increase the accumulation of starch within source leaves of soybean (Huber et al., 1984; Sharkey et al., 1985).
In leaves of tomato under elevated C02, basal (morning) leaf starch content was higher and the daily change greater due to a prolonged duration of starch accumulation during the day (Yelle et al., 1989). Leaves of bean (Hoddinot and Jolliffe, 1988) and soybean (Allen et al., 1988) also have an increased basal starch content. These findings suggested that increasing concentration of CO2 altered the diel pattern of deposition and mobilization of chloroplast starch.
Such increases in leaf starch may be the cause of the increase in specific leaf weight often observed under
conditions of elevated CO2 (Ehret and Jollife, 1985). Another example is soybean which was reported to have greater specific leaf weight (SLW) in elevated-C02-grown plants (Allen et al., 1988; Campbell et al., 1988), while basil (Ocimum basilicum) was observed to exhibit a 1.5 to 2-fold greater increase in SLW as a result of an increase in atmospheric C02 concentration from 400 to 1500 UL C02 L' (Holbrook et al.,




52
1993). At 1500 IlL CO2 L starch accounted for up to 38% of the total SLW of basil compared to <10% of SLW of spinach leaves. Furthermore, basil leaves grown in enriched-C02 showed chlorotic symptoms, whereas spinach leaves did not exhibit chlorosis. Arp (1991) proposed that starch accumulation appears to be primarily a symptom of the imbalance between supply and demand of carbohydrates and does not represent a significant feedback mechanism on photosynthetic capacity.
Plants that do not form large amounts of starch tend to accumulate sucrose during the day; however those species such as soybean that accumulate starch typically do not accumulate soluble sugar (Allen et al., 1988; Huber, 1989).
Starch and soluble sugar levels in leaves of wild radish plants (Raphanus sativus x raphanistrum) increased with increasing atmospheric [C02], whereas specific leaf area and nitrogen concentration of leaves significantly decreased (Chu et al., 1992). Fructose and glucose accumulated to a greater extent than sucrose at high C02 and may have been utilized for synthesis of cell-wall components, contributing to higher specific leaf weight. The ratio of starch to sucrose
concentration of soybean leaves (Allen et al., 1988) and rice leaves (Rowland-Bamford et al., 1990) was positively correlated with the CO2 concentration. Similarly, the starch:sucrose ratio of bean leaves also showed a marked increase with increasing intercellular CO2 partial pressure




53
(Sharkey et al., 1985). Moreover, formation of leaf starch and sucrose of bean was linearly related to assimilation. Due to the higher level of assimilate buildup and lower stomatal conductance to water loss, crops grown under high C02 may have capacity to delay stress effects on metabolism for a few days (Allen, 1994).
Water-stressed leaves have decreased rates of starch synthesis and increased synthesis of sugars (Morgan, 1984). The author sugested that it is possible that changes in enzyme activities of the pathway leading to starch synthesis play a substantial role in altering partitioning during stress.
When attached, translocating P. vulgaris leaves were water stressed and partitioning was assayed by 10-min pulselabelling with 14CO2, starch accumulation was decreased by more than 75% (Vassey and Sharkey, 1989).
The purpose of this research was to investigate the effects of elevated atmospheric CO2 and water stress during panicle initiation and anthesis stages on sucrose, starch and fructose concentration of rice plant leaves.
Materials and Methods
Plant material. Plant material was collected from
rice plants as described in Chapter 2. For carbohydrate analysis, fully expanded leaves were sampled at 12:00 noon eastern standard time on various dates before drought, during drought and after re-watering. From each chamber, half the




54
leaves were collected from the eastern side and half from the
western side. The dry weight was determined after oven-drying at 701C for 48 hours. Oven-dried leaves were ground in a ball mill to a fine powder.
Sugar and starch analysis. Sucrose and starch
determinations were carried out as described by Boote (1974) with slight modification. Oven-dried powdered leaf material
(0.05 g) was extracted four times with 3 ml of 80% ethanol (v/v) at 950C for 1-h. It was confirmed that this was sufficient to quantitatively extract the ethanol-soluble sugars in the plant material. After centrifugation, supernatants were pooled and brought to a total volume of 15
ml with 80% ethanol. Sucrose, as fructose equivalents in sucrose, was analyzed with the resorcinol method described by
Roe (1934) with some modification. A 0.25-mL aliquot was brought to 1 ml with 0.75 mL of distilled water and then boiled for 10 min with 1 mL of 1N NaOH to destroy free fructose. After cooling, 1 mL resorcinol (0.1%, w/v in 95% ethanol) and 7 ml of HCl O9N) were added, mixed and the tubes incubated at 800C for 8 min. Samples were cooled and read in spectrophotometer at 520 nm. Total fructose equivalents (free fructose plus fructose equivalents in sucrose) were determined by the resorcinol test as described above except samples were
boiled with 1 mL distilled water instead of 1N NaOH. Free fructose was calculated from the difference between total fructose equivalents minus fructose equivalents of sucrose and




55
reported after multiplying by 0.5263 (molar ratio of fructose to sucrose) because sucrose was used as the standard.
The pellet obtained after 80% ethanol extraction was dried overnight at 60'C. Four ml of phosphate buffer was added to the dried pellet. In addition, a plant sample standard, starch standards, and a blank were similarly treated. After adding 1 ml of dialyzed a-amylase, the test tubes were incubated at 85'C for 1 h. After adding 5 ml of acetate buffer and 1 ml of enzyme mix (containing: amyloglucosidase, invertase, acetate buffer and water) the test-tubes were incubated in a shaking water bath at 48C for 24 h. Following incubation, the samples were filtered and the glucose content in the supernatant was determined by the Nelson-Somogyi test (Spiro, 1966). The results were converted to starch equivalents by comparing standard starch samples and glucose standards run in parallel with the plant material.
Results and Discussion
Leaf sucrose concentration. Data of leaf sucrose concentration (mg g' DW) of rice grown at 350 and 700 1 CO2 1-' and under four different water management are given in Table 3.1 through 3.3. Effects of [C02] were significantly different throughout the season. Water stress treatments were significantly different in sucrose level on dates when water deficit occurred. As described earlier, drought stress at panicle initiation was begun at 57 DAP; therefore drought-




56
stress was still absent and only [C0] effects were present at 55 and 58 DAP. The average increase in leaf sucrose
concentration of double- [CO2] plants from 55 DAP through the end of season was 9% under the continuously flooded treatment.
Increase in leaf sucrose concentration of high-CO2 plants has been also reported in soybean plants (Allen et al., 1988; Sicher et al., 1995), and increases of 40% were reported for rice (Rowland-Bamford et al., 1990).
By 72 DAP (Table 3.1), 15 days after flood water was drained to initiate water stress, water management treatments were significantly different and showed [C02] effects as well as the interaction of both water management and [C02]. By 74 DAP, plants for 350DPI and 350DBS treatments started leaf curling/desication and sucrose concentration of ambient-C2 plants decreased by 82% (Table 3.1). Due to ability of highCO2 to maintain high leaf water potential, 700DPI and 700DBS treatments had higher sucrose concentration compared to 350[C02] plants. At this time, both the 350DPI and 350DPA treatments had been re-watered for 1 day, so that sucrose concentration increased at 75 DAP, whereas 700DPI AND 700DBS plants had reached their leaf curling/desication point on this day (re-watered after sampling on 75 DAP) and water deficit decreased leaf sucrose concentration of elevated-C02 plants by 86% (Table 3.2).
At 100 DAP (Table 3.3), there were no significant water deficit effects on leaf sucrose concentration,but effects were




57
significant at 109 DAP (P<0.01). Leaf sucrose concentration of 350DAN and 700DAN treatments was lower than the concentration of 350DBS and 700DBS treatments (Table 3.3), possibly because DBS treatments senesed lower leaves during the previous drought cycle at panicle initiation, and had less transpiring surface (Baker et al., 1996a, 1996b). Minor
effects of water deficit during anthesis carried through to 125 DAP (P<0.01). Decreases in sucrose concentration of stressed leaves by 69% in low-CO2 and 58% in doubled-C02 plants were observed for DAN water deficit treatments. However, sucrose concentration was decreased only by 37% in low-CO2 and 32% in elevated-C02 plants for rice that was exposed to water deficit at two stages of growth (DBS).
Leaf starch concentration. Leaf starch concentration (mg g 1 DW) of rice plants grown at 350 and 700 JIL CO2 L-1 and four different water regimes are given in Table 3.4 through 3.6. The [C021 effects on leaf starch were significant during early reproductive stages (58 through 74 DAP) (P<0.05). Elevated-C02 plants had higher starch concentration before and after panicle initiation which occurred at 74 DAP, but after that starch concentration of high-CO2 and ambient-CO2 plants were similar.
Increase in leaf starch concentration of elevated-C2 plants has also been reported for soybean (Allen et al., 1988), bean (Radaglou and Jarvis, 1992) and rice (RowlandBamford et al., 1990). The difference in leaf starch and




58
sucrose concentration between leaves grown at elevated and current CO2 diminished with plant maturity.
Water stress effects on starch concentration were significant at 72, 74, 75, 82, and 109 DAP as shown in Table 3.4 through 3.6. The CO2 by water management interaction was significantly different only at 75 DAP. After paddy was drained from chambers (350DPI, 350DBS, 700DPI and 700DBS) at
57 DAP for stress treatment at panicle initiation, a decrease in leaf starch concentration was detected at 74 DAP (Table 3.4). Low-CO2 plants (350DPI) were more quickly affected by water deficit treatment so that leaf starch concentration of that treatment decreased by 93%. By 75 DAP, leaves of highCO2 plants were highly stressed and leaf starch concentration decreased by 92%.
After water was restored to the drought treatment at 74
and 75 DAP for ambient-CO2 and elevated-CO2, respectively, the stressed plants of both [C021 treatments recovered to initial starch value by 89 DAP (Table 3.8), so that there was no significant difference in water-treatment on that day. It is
interesting to note that water -deficit -induced differences in leaf starch persisted for more than 7 days after water was restored (until at least 82 DAP).
For stress treatment at anthesis, paddy water was drained from chambers at 93 DAP in treatments 350DAN and 700DAN, and at 96 DAP in 35ODBS and 700DBS. At 100 DAP (Table 3.5), the
leaf starch concentration was not affected by water stress




59
treatment (P=0.11). By 109 DAP (Table 3.9), the decrease in leaf starch concentration was significant, and the water management treatments were significantly different (P<0.01).
Decrease in leaf starch concentration due to water
deficit has also been observed by Morgan (1984) and Vassey and Sharkey (1989). The later authors found 75% decrease in leaf starch concentration in bean (Phaseolus vulgaris).
The decrease in leaf starch and sucrose of plants with increasing plant development has been observed on rice (Rowland-Bamford et al., 1990) and wheat (Nie et al., 1995).
Decline in leaf starch and sucrose concentration might be associated with the onset of grain filling.
Leaf fructose concentration. Leaf fructose
concentrations in leaves from rice grown at 350 and 700 iUl CO2 1' and in various water management treatments are listed in Table 3.7 through 3.9. Leaf fructose concentration of C02enhanced plants were significantly higher compared to those grown at ambient CO2 treatment throughout the season (P<0.01) Leaf fructose concentration of plants grown in high-CO2 chambers under continuously flooded treatment was 28 to 92%
higher than ambient-CO2 plants for the various dates from panicle initiation (54 DAP) through to final harvest (127 DAP) Elevated CO2 treatments had relatively higher fructose concentration early in the season (DAP 55 and 58) than later (DAP 72 and beyond).




60
Water stress effects were significant at 74, 75, 82 and 109 DAP (Table 3.7, 3.8, and 3.9). At 74 and 109 DAP,
elevated-CO2 plants were able to maintain leaf fructose concentration relatively higher than those of low-CO2 plants. After paddy water was drained from chambers (350DPI, 350DBS, 700DPI and 700DBS) at 57 DAP for stress treatment at panicle initiation, a decrease in leaf fructose concentration was detected at 74 DAP, and at that time, elevated-CO2 plants were able to maintain leaf fructose concentration relatively higher than ambient-C02 plants.
After water was restored at 74 and 75 DAP for ambient-CO2 and elevated-C02 treatments, respectively, stressed plants of both [CO2] treatments recovered to initial fructose concentration values by 92 DAP (Table 3.8). There was no significant effect of water-management treatment at 92 DAP.
For stress treatment at anthesis, paddy water was drained from chambers at 93 DAP for treatments of 350DAN and 700DAN, and at 96 DAP for 350DBS and 700DBS treatments. At 100 DAP (Table 3.9), the leaf fructose concentration was not affected by water stress treatment (P=0.82), however by 109 DAP (Table 3.9), the decrease in leaf fructose was significant for the water treatment (P<0.01). Leaves of plants grown in high-CO2 were able to maintain higher leaf fructose concentration and were able to recover more quickly than those of plants grown at low CO2.




61
Elevated CO2 treatments were able to avoid or compensate for water stress for 1 to 2 days longer, i.e., elevated-CO2 plants were able to maintain moderate sucrose, starch and fructose concentration 1 to 2 days longer compared to ambientCO2 plants. Elevated-CO2 plants had ability to recover more quickly than ambient-CO2 plants.




62
Table 3.1. Leaf sucrose concentration of rice plants grown at 350 and 700 IiL CO2 L 1 and in four different water managements. Leaf samples were taken at noon eastern standard time from 55, 58, 72 and 74 day-old plants.
CO2 Water Leaf Sucrose Concentration
Management 55DAP 58DAP 72DAP 74DAP
iL L' mg g' dry weight
350 FLD 82.04.0 73.18.7 66.72.6 62.63.8
DPI 84.22.7 73.74.2 49.13.0 9.42.9
DAN 83.34.5 74.66.0 66.24.6 63.40.7
DBS 85.35.0 75.64.6 53.55.3 12.63.5
700 FLD 95.01.2 85.33.4 80.91.9 78.31.9
DPI 92.81.4 87.42.5 58.74.1 28.31.7
DAN 93.43.2 86.44.6 80.82.3 74.42.6
DBS 90.05.0 85.56.6 53.84.9 28.31.2
CO2 P=0.0001 P=0.0001 P=0.0001 P=0.0001
WM P=0.9744 P=0.9658 P=0.0001 P=0.0001
CO2*WM P=0.3071 P=0.9413 P=0.0170 P=0.7301
Note:
FLD: continuously flooded DPI: drought imposed during panicle initiation DAN: drought imposed during anthesis DBS: drought imposed during both stages DAP: days after planting WM : water management
Paddy water was removed at 57 DAP to initiate water stress at panicle initiation in 350DPI, 350DBS, 700DPI and 700DBS treatments.
water was restored after sampling at 74 DAP in 350DPI and 350DBS treatments, and at 75 DAP in 700DPI and 700DBS treatments.




63
Table 3.2. Leaf sucrose concentration of rice plants grown at 350 and 700 pL CO2 L I and in four different water managements. Leaf samples were taken at noon eastern standard time from 75 (stressed), 82 and 89 (recovery) day-old plants. Treatments 350DPI and 350DBS had been re-watered for 1 day, but 700DPI and 700DBS had not yet been watered.
CO2 Water Leaf Sucrose Concentration
Management 75DAP 82DAP 89DAP
IL L' mg g dry weight
350 FLD 59.62.3 59.22.4 59.51.3
DPI 28.72.8 51.02.2 61.11.4
DAN 59.01.9 58.21.8 58.92.3
DBS 22.22.6 52.82.0 64.51.4
700 FLD 65.91.6 60.61.5 58.11.9
DPI 9.61.6 51.92.4 63.71.7
DAN 68.72.6 60.31.8 59.62.8
DBS 7.72.8 52.61.4 63.02.6
-------------------------------------------------C02 P=0.0530 P=0.6008 P=0.9325
WM P=0.0001 P=0.0162 P=0.1256
C02*WM P=0.0001 P=0.9824 P=0.7765
Note:
FLD: continuously flooded DPI: drought imposed during panicle initiation DAN: drought imposed during anthesis DPA: drought imposed during both stages DAP: days after planting WM : water management
Water was restored after sampling at 74 DAP in 350DPI and 350DBS treatments, and at 75 DAP in 700DPI and 700DBS treatments.




64
Table 3.3. Leaf sucrose concentration of rice plants grown at 350 and 700 IiL C02 L' and in four different water managements. Leaf samples were taken at noon eastern standard time from 100, 109 (deficit period) and 125 (recovery period) day-old plants.
C02 Water Leaf Sucrose Concentration
Management 100DAP 109DAP 125DAP
UL L' mg g dry weight
350 FLD 47.92.1 50.31.2 39.10.8
DPI 51.60.9 48.61.2 41.80.8
DAN 50.80.7 15.51.0 35.41.2
DBS 51.00.6 31.70.9 36.31.3
700 FLD 50.21.4 44.61.3 37.42.2
DPI 50.40.6 48..41.1 40.50.7
DAN 48.50.6 18.90.8 34.31.2
DBS 51.51.2 30.51.8 34.51.1
----------------------------------------..-------CO2 P=0.8763 P=0.0550 P=0.0105
WM P=0.2230 P=0.0001 P=0.0001
CO2*WM P=0.2629 P=0.0001 P=0.0537
Note:
FLD: continuously flooded DPI: drought imposed during panicle initiation DAN: drought imposed during anthesis DPA: drought imposed during both stages DAP: days after planting WM : water management
Paddy water was removed at 93 DAP in 350DAN and 700DAN treatments, and at 96 DAP in 350DBS and 700DBS treatments. Water was restored after sampling at 110 DAP in 350DAN and 700DAN treatments, and at 111 DAP in 350DBS and 700DBS treatments.




65
Table 3.4. Leaf starch concentration of rice plants grown at 350 and 700 I.L CO2 L and in four different water managements. Leaf samples were taken at noon eastern standard time from 55, 58, 72 and 74 day-old plants.
CO2 Water Leaf Starch Concentration
Management 55DAP 58DAP 72DAP 74DAP
L L-' mg g dry weight
350 FLD 54.52.0 42.31.8 53.82.6 46.31.2
DPI 56.71.9 43.31.9 37.43.0 3.40.4
DAN 55.31.5 41.51.0 55.33.1 47.31.5
DBS 51.42.2 44.41.2 33.42.4 3.62.5
700 FLD 76.82.4 59.32.4 59.51.8 51.11.2
DPI 78.91.4 61.42.5 54.42.8 10.31.4
DAN 71.92.3 61.11.7 57.71.3 47.01.3
DBS 75.41.4 63.31.3 37.71.7 11.71.5
-------------------------------------------------CO2 P=0.0001 P=0.0001 P=0.0030 P=0.0024
WM P=0.5909 P=0.7728 P=0.0001 P=0.0001
C02*WM P=0.7419 P=0.9787 P=0.1037 P=0.1683
Note:
FLD: continuously flooded DPI: drought imposed during panicle initiation DAN: drought imposed during anthesis DBS: drought imposed during both stages DAP: days after planting WM : water management
Paddy water was removed at 57 DAP to initiate water stress at panicle initiation in 350DPI, 350DBS, 700DPI and 700DBS treatments.
Water was restored after sampling at 74 DAP in 350DPI and 350DBS treatments, and at 75 DAP in 700DPI and 700DBS treatments.




66
Table 3.5. Leaf starch concentration of rice plants grown at 350 and 700 IiL CO2 LI and in four different water managements. Leaf samples were taken at noon eastern standard time from 75 (stressed), 82 and 89 (recovery) day-old plants. Treatments 350DPI and 350DBS had been re-watered for 1 day, but 700DPI and 700DBS had not yet been watered.
CO2 Water Leaf Starch Concentration
Management 75DAP 82DAP 89DAP
UL LI' ----- mg g1 dry weight
350 FLD 40.11.5 21.01.5 21.71.9
DPI 10.32.0 12.81.9 21.40.9
DAN 40.71.3 25.32.2 20.11.3
DBS 8.42.1 11.42.0 22.81.0
700 FLD 50.72.0 24.72.1 21.80.7
DPI 4.12.3 13.51.3 21.50.8
DAN 50.32.6 22.61.8 21.70.9
DBS 7.52.8 14.32.8 20.21.6
CO2 P=0.0860 P=0.4175 P=0.7964
WM P=0.0001 P=0.0001 P=0.9048
CO2*WM P=0.0103 P=0.4020 P=0.3998
Note:
FLD: continuously flooded DPI: drought imposed during panicle initiation DAN: drought imposed during anthesis DPA: drought imposed during both stages DAP: days after planting WM : water management
Water was restored after sampling at 74 DAP in 350DPI and 350DBS treatments, and at 75 DAP in 700DPI and 700DBS treatments.




67
Table 3.6. Leaf starch concentration of rice plants grown at 350 and 700 iL CO2 L and in four different water managements. Leaf samples were taken at noon eastern standard time from 100, 109 (stressed) and 125 (recovery) day-old plants.
C02 Water Leaf Starch Concentration
Management 100DAP 109DAP 125DAP
pL L'- mg g' dry weight
350 FLD 11.51.4 11.80.9 12.51.4
DPI 11.81.6 11.71.2 12.71.0
DAN 10.90.9 7.00.3 11.30.9
DBS 12.70.5 9.40.7 10.01.5
700 FLD 13.41.2 12.10.9 11.31.1
DPI 12.91.5 10.30.4 12.22.3
DAN 10.41.8 7.81.6 11.21.0
DBS 12.81.5 10.62.0 12.11.6
-------------------------------------------------CO2 P=0.2979 P=0.6194 P=0.9207
WM P=0.1135 P=0.0001 P=0.6042
C02*WM P=0.5881 P=0.2438 P=0.5021
Note:
FLD: continuously flooded DPI: drought imposed during panicle initiation DAN: drought imposed during anthesis DPA: drought imposed during both stages DAP: days after planting WM : water management
Paddy water was removed at 93 DAP in 350DAN and 700DAN treatments, and at 96 DAP in 350DBS and 700DBS treatments. Water was restored after sampling at 110 DAP in 350DAN and 700DAN treatments, and at 111 DAP in 350DBS and 700DBS treatments.




68
Table 3.7. Leaf fructose concentration of rice plants grown at 350 and 700 IiL CO2 L' and in four different water managements. Leaf samples were taken at noon eastern standard time from 55, 58, 72 and 74 day-old plants.
CO2 Water Leaf Fructose Concentration
Management 55DAP 58DAP 72DAP 74DAP
IL L' mg g' dry weight
350 FLD 5.01.2 4.91.7 8.80.8 9.20.8
DPI 4.21.3 4.60.9 5.01.6 0.91.9
DAN 4.50.4 5.00.5 8.70.8 9.21.9
DBS 4.60.5 4.70.7 4.71.3 1.10.7
700 FLD 8.20.3 9.41.3 11.31.2 11.91.2
DPI 8.31.3 9.52.5 11.81.5 8.91.6
DAN 8.40.5 9.00.5 11.61.6 12.51.2
DBS 8.21.2 9.21.6 12.70.8 7.91.7
CO2 P=0.0001 P=0.0001 P=0.0001 P=0.0001
WM P=0.2252 P=0.9928 P=0.0139 P=0.0001
C02*WM P=0.2296 P=0.5426 P=0.0007 P=0.0001
Note:
FLD: continuously flooded DPI: drought imposed during panicle initiation DAN: drought imposed during anthesis DBS: drought imposed during both stages DAP: days after planting WM : water management
Paddy water was removed at 57 DAP to initiate water stress at panicle initiation in 350DPI, 350DBS, 700DPI and 700DBS treatments.
Water was restored after sampling at 74 DAP in 350DPI and 350DBS treatments, and at 75 DAP in 700DPI and 700DBS treatments.




69
Table 3.8. Leaf fructose concentration of rice plants grown at 350 and 700 IjL CO2 L and in four different water managements. Leaf samples were taken at noon eastern standard time from 75 (stressed), 82 and 89 (recovery) day-old plants. Treatments 350DPI and 350DBS had been re-watered for 1 day, but 700DPI and 700DBS had not yet been watered.
CO2 Water Leaf Fructose Concentration
Management 75DAP 82DAP 89DAP
IL L' mg g' dry weight
350 FLD 8.60.5 9.22.4 8.00.4
DPI 3.20.8 5.60.5 8.91.4
DAN 8.91.9 8.50.7 8.90.8
DBS 2.81.6 6.41.5 7.51.4
700 FLD 11.91.6 12.21.7 11.01.9
DPI 1.91.8 9.71.0 12.01.7
DAN 13.10.7 12.31.4 11.90.9
DBS 1.61.8 9.60.6 12.41.2
CO2 P=0.0034 P=0.0001 P=0.0001
WM P=0.0001 P=0.0001 P=0.6729
C02*WM P=0.0001 P=0.4380 P=0.0302
Note:
FLD: continuously flooded DPI: drought imposed during panicle initiation DAN: drought imposed during anthesis DPA: drought imposed during both stages DAP: days after planting WM : water management
Water was restored after sampling at 74 DAP in 350DPI and 350DBS treatments, and at 75 DAP in 700DPI and 700DBS treatments.




70
Table 3.9. Leaf fructose concentration of rice plants grown at 350 and 700 VL CO2 L' and in four different water managements. Leaf samples were taken at noon eastern standard time from 100, 109 (stressed) and 125 (recovery) day-old plants.
CO2 Water Leaf Fructose Concentration
Management 100DAP 109DAP 125DAP
4L L-' ------- mg g' dry weight -------350 FLD 8.11.2 7.71.2 7.00.8
DPI 9.01.9 7.80.4 6.90.8
DAN 8.30.7 1.51.0 6.90.2
DBS 8.90.6 3.80.9 6.71.3
700 FLD 10.21.4 9.61.3 7.91.2
DPI 10.51.6 9.41.1 7.60.7
DAN 10.71.6 5.30.8 7.71.2
DBS 10.51.2 7.70.3 7.61.1
CO2 P=0.0001 P=0.0001 P=0.0001
WM P=0.8181 P=0.0001 P=0.2932
C02*WM P=0.9037 P=0.0039 P=0.9681
Note:
FLD: continuously flooded DPI: drought imposed during panicle initiation DAN: drought imposed during anthesis DPA: drought imposed during both stages DAP: days after planting WM : water management
Paddy water was removed at 93 DAP in 350DAN and 700DAN treatments, and at 96 DAP in 350DBS and 700DBS treatments. Water was restored after sampling at 110 DAP in 350DAN and 700DAN treatments, and at 111 DAP in 350DBS and 700DBS treatments.




CHAPTER 4
THE EFFECTS OF ELEVATED CO2 CONCENTRATION AND WATER STRESS ON SUCROSE PHOSPHATE SYNTHASE ACTIVITY
Introduction
Sucrose phosphate synthase (SPS) is a key enzyme in the rate-control of sucrose synthesis and its activity in a variety of species has been negatively correlated with leaf starch accumulation (Huber, 1981, 1983; Huber et al., 1984).
Negative correlation between SPS activity and starch accumulation for C02-enriched plants has observed for leaves of Cucumis sativus (Peet et al., 1986). Short-term treatment of soybean plants with elevated CO2 doubled the starch content and decreased the activity of SPS within the leaves (Huber et al., 1982, 1984). In contrast, the SPS activity of rice leaves measured 59 d after planting in 660 jjL L' C02 has been found to increase two- and three-fold when compared with the control (Hussain et al., 1990).
Moreover, soybean plants grown in high-N media (20 mol mKNO3) have a higher rate of assimilate export, higher SPS activity, higher sucrose and decreased starch concentration within the leaves when compared to plants grown at 10 mol m-3 KNO3 (Huber et al., 1984). Short-term treatment of high-N supplied plants with elevated CO2 increased leaf starch 71




72
concentration at the expense of export of assimilate from the leaf. Although the sucrose concentration of the leaf in this case was unchanged, there was a marked reduction of SPS activity (Huber et al., 1984).
SPS plays an important role as the limiting factor of partitioning of carbon sources. Therefore, it seems that enhancement of SPS activity may promote the. ability of the source function.
Makino et al. (1994) demonstrated that CO2 assimilation rates of young rice plants grown at ambient CO2 levels were limited by SPS activity when blades were exposed to high CO2 during gas-exchange measurements. When plants are exposed continuously to high C02, synthesis of SPS may be increased to optimize photosynthesis and growth under this particular environment, thereby resulting in greater SPS activity.
C02 enrichment increased SPS activity of the rice cultivar IR-30 when measurements were made at the midtillering phase (Baker et al., 1988). In the same study, it was demonstrated that CO2 enrichment had no effect on SPS activity of soybean leaves. In contrast to rice, soybean leaves accumulate large amounts of starch (Huber et al., 1982, 1984; Allen et al., 1988).
The C02-saturated photosynthesis was positively correlated with the activities of SPS in the high-N leaves. SPS activity increased steadily with increasing leaf nitrogen concentration (Makino et al., 1994).




73
Seneewera et al. (1995) concluded that the enhancement of SPS activity by CO2 enrichment in fully expanded leaf blades plays a major role in determining the capacity of these source blades to supply the growing sink with sucrose.
In short term water deficit, the partitioning of newlyfixed photosynthate favouring sucrose at the expense of starch is among the early effects of tissue dehydration in some species (Quick et al., 1989, 1992). This increased partitioning of recent assimilates to sucrose has been linked to alterations in sucrose phosphate synthase (SPS) activity and to a general increase in the amounts of metabolites due to a decrease in cytoplasmic volume under drought (Quick et al., 1989). Although some disagreements have been observed as a consequence of measurements being made with plants either under ambient or saturating C02 (Vassey and Sharkey, 1989; Vassey et al., 1991), it is likely that the preferential partitioning of recent assimilates to sucrose in waterstressed leaves is accompanied by an increased activation of SPS in spinach (Zrenner and Stitt, 1991).
Little is known about the effect of enhanced C02 concentration and water stress during panicle initiation and anthesis on the activities of sucrose phosphate synthase in leaves of rice. Therefore this study was conducted to evaluate possible interaction effects of elevated CO2 and water stress on SPS activity of rice around panicle initiation and anthesis stages.




74
Materials and Methods
Plant materials. Plant material was collected from rice plants grown under conditions described in Chapter 2. For measurements of SPS activity, 15 fully-expanded leaves were excised and rapidly immersed in liquid N2 around 12:00 noon eastern standard time on various dates before drought, during drought and after re-watering. There were 8 sunlit, controlled-environment chambers of which 4 chambers were set at 350 pL CO2 L' and 4 chamber were set at 700 liL CO2 L-1. In each pair of chambers (one ambient and one elevated [CO2]), the following water management-stress regimes were imposed: a) continuously flooded (FLD), b) paddy flood water removed and drought stress imposed during panicle initiation (DPI), c) drought stress imposed during anthesis (DAN), and d) drought stress imposed during both panicle initiation and anthesis (DBS). Leaves were collected from both the eastern half and the western half of each chamber and composited prior to storage in liquid N.
Enzyme extraction. Leaf samples previously frozen and stored in liquid nitrogen were weighed (0.2 g) and ground in chilled mortar with 2.5 ml extraction buffer (50 mM MOPS-NaOH [pH 7.5], 15 mM MgC2, 1 mM EDTA, 2.5 mM DTT, and 0.1% (v/v) Triton X-100). The extract was centrifuged in a 1.2-ml microtube at 13,000 g for 1.5 min. The supernatant solution was desalted by centrifugal gel filtration into buffer




75
containing 50 mM MOPS-NaOH [pH 7.5], 15 mM MgCl2, 1 mM EDTA, and 2.5 mM DTT.
Enzyme Assay. Sucrose phosphate synthase was measured in the synthetic direction by quantitation of sucrose formation using the resorcinol method. Assays were conducted by
incubating 45 pL of tissue extract for 10 min at 25 'C with 10 mM UDPG, 10 mM F6P, 40 mM G6P, 50 mM MOPS-NaOH (pH 7.5), 15 mM MgCl2 and 2.5 mM DTT in a total volume of 70 pL. The reaction was terminated by the addition of 70 pL 1 N NaOH. Tubes were boiled for 10 min to destroy any remaining fructose (or unreacted F6P), then 250 UL 0.1% (w/v) resorcinol in 95% ethanol plus 750 iL 30% (v/v) HC1 were added. Tubes were incubated at 80'C for 8 min and after cooling, the As2o was measured with a Spectrophotometer. Sucrose formation was quantitated by comparison to a sucrose standard curve after subtraction of Ao20 at 0 min (background). The background standard was handled essentially the same as described earlier except that 70 IL 1N NaOH was added to reaction mixture before adding extract tissue, so that the reaction would not occur.
Results and discussion
Sucrose phosphate synthase. Sucrose phosphate synthase activities in leaves from rice grown at 350 and 700 UL C02 L1 and in various water management treatment are listed in Table 4.1 and 4.2. SPS activities of CO2-enhanced plants were significantly higher compared to those grown at ambient CO2




76
treatment throughout the season (P<0.05). SPS activities of plants grown in high C02 chambers under continuously-flooded treatment were consistenly higher than ambient CO2 treatments by 6 to 16% from panicle initiation (54 DAP) through right before final harvest (127 DAP).
The results obtained in this study indicate that CO2 enrichment increased SPS activities throughout the season in rice leaves when measured under continuously-flooded water management. Enhancement of SPS activity by elevated CO2 levels has also been reported on rice (Baker et al., 1988; Hussain et al., 1990; Makino et al., 1994; and Seneweera et al., 1995). Huber et al. (1984), however, demonstrated that CO2 enrichment had no effect on SPS activity of soybean leaves.
Water stress effects were significant at 74 and 110 DAP (Table 4.1 and 4.2). On those dates, elevated-CO2 plants were able to maintain SPS activity relatively higher than those of low-CO2 plants. After paddy water was withdrawn from chambers (350DPI, 350DBS, 700DPI and 700DBS) at 57 DAP for stress treatment at panicle initiation, a decrease in SPS activity was detected at 74 DAP (Table 4.1). Water management
treatments were significantly different in SPS activities at 74 and 110 DAP (P<0.001).
After water was restored for DPI and DBS treatments at 74 and 75 DAP for ambient-C02 and elevated-C02, respectively, plants of both [C021 treatments recovered to initial SPS activities by 82 and 92 DAP (Table 4.1), so that there was no




77
significant difference of water-treatment on those recovery days.
For stress treatment at anthesis, paddy water was drained from chambers at 93 DAP for treatments of 350DAN and 700DAN, and at 96 DAP for 350DBS and 700DBS treatments. At 100 DAP (Table 4.2), the effect of water stress treatment was not yet significantly different (P=O.57). A decrease in SPS activity was detected at 110 DAP, when the water deficit treatment was significantly lower (P<0.01).
Studies of water stress effects on sucrose phosphate synthase (SPS) activities have also been studied on soybean (Quick et al., 1989, 1992), Phaseolus vulgaris (Vassey and Sharkey, 1989; Vassey et al., 1991), and spinach (Zrenner and Stitt, 1991). However, they have failed to reach a consensus (Quick et al., 1989; Vassey and Sharkey, 1989). In soybean, water-stressed leaves had SPS activities that were not significantly different from controls, thus this element of export capacity was maintained. However, in Phaseolus
vulgaris leaves, mild water stress (total leaf water potential at about -1.0 MPa) inhibited SPS by more than 60% (Vassey and Sharkey, 1989).
A study of spinach leaves showed that water stress had differing effects on two kinetic forms of SPS, detected by assay conditions (Quick et al., 1989). with "nonselective" assay conditions, where substrate concentration was high and orthophosphate (Pl) was omitted, there was no significant




78
effect of water stress on SPS activity. With "selective" assay conditions, where Pi was included to inactivate the kinetically-inactive form of the enzyme and reveal the kinetically-active form, a substantial increase in SPS
activity was found as total leaf water potential was decreased from 0 to -1.2 MPa. The previously mentioned studies of soybean and P. vulgaris used assay conditions that were nonselective. Thus, the extent to which changes in SPS activity contribute to changes in partitioning between sucrose and starch may depend on the relative contribution of these kinetic forms to SPS activity as it exists in vivo.




79
Table 4.1. Sucrose phosphate synthase activities of rice plants grown at 350 and 700 IjL CO2 Ll and in four different water managements. Plant samples were taken at noon eastern standard time from 54, 74 (stressed), 82 and 92 (recovery) day-old plants.
CO2 Water SPS Activities
Management 54DAP 74DAP, 82DAP-- 92DAP
pL L' --- pmol (g fresh weight)' h- --350 FLD 49.70.1 47.63.5 47.11.4 44.41.2
DPI 50.20.9 13.41.3 46.61.7 44.83.9
DAN 50.21.7 48.63.6 47.11.4 44.92.3
DBS 50.53.5 15.34.1 46.61.7 45.83.1
700 FLD 55.20.7 54.82.1 51.30.1 49.23.8
DPI 55.22.5 23.24.5 50.04.8 49.00.6
DAN 54.83.4 62.33.6 50.83.6 49.71.7
DBS 55.40.4 21.94.2 51.00.8 49.50.1
CO2 P=0.0014 P=0.0047 P=0.0126 P=0.0082
WM P=0.9866 P=0.0001 P=0.9615 P=0.9608
C02*WM P=0.9882 P=0.6898 P=0.9904 P=0.9852
Note:
FLD: continuously flooded DPI: drought imposed during panicle initiation DAN: drought imposed during anthesis DBS: drought imposed during both stages DAP: days after planting WM : water management
Paddy water was removed at 57 DAP to initiate water stress at panicle initiation in 350DPI, 350DBS, 700DPI and 700DBS treatments.
Water was restored after sampling at 74 DAP in 350DPI and 350DBS treatments, and at 75 DAP in 700DPI and 700DBS treatments.




80
Table 4.2. Sucrose phosphate synthase activities of rice plants grown at 350 and 700 IlL CO2 LI and in four different water managements. Plant samples were taken at noon eastern standard time from 100, 110 (stressed) and 127 (recovery) dayold plants.
CO2 Water SPS Activities
Management 100DAP 1IODAP 127DAP
UL L' --- Umol (g fresh weight)' h-' --350 FLD 41.61.8 38.63.4 28.41.1
DPI 42.20.9 39.83.4 27.71.4
DAN 40.61.9 15.01.0 28.12.3
DBS 41.22.5 25.41.7 27.72.2
700 FLD 44.80.6 45.81.3 34.82.9
DPI 44.91.3 46.54.4 32.23.2
DAN 42.32.7 15.51.1 32.70.0
DBS 43.62.8 26.91.5 30.63.4
CO2 P=0.0303 P=0.0114 P=0.0044
WM P=0.5696 P=0.0001 P=0.5426
CO2*WM P=0.9848 P=0.1890 P=0.7804
Note:
FLD: continuously flooded DPI: drought imposed during panicle initiation DAN: drought imposed during anthesis DBS: drought imposed during both stages DAP: days after planting WM : water management
Paddy water was removed at 93 DAP in 350DAN and 700DAN treatments, and at 96 DAP in 350DBS and 700DBS treatments. Water was restored after sampling at 110 DAP in 350DAN and 700DAN treatments, and at 111 DAP in 350DBS and 700DBS treatments.




CHAPTER 5
COORDINATION OF CARBON METABOLISM ACTIVITIES
This chapter briefly describes the relationship between carbohydrate levels and SPS activity from data cited in previous chapters 2, 3 and 4.
Increasing CO2 increased sucrose and starch concentration and also SPS activity. The increase in sucrose concentration and SPS activity was relatively greater compared to starch concentration throughout the season (Figures 5.1 and 5.2). For continuously-flooded plants, SPS activity, sucrose and starch concentration were initially high and decreased with
increasing plant maturity. Starch concentration declined earlier compared to SPS activity and sucrose concentration (Figures 5. 1) A considerable decrease in starch was observed during the end of growing season, while sucrose concentration and SPS activity was maintained moderately high. The decrease in sucrose and starch concentration in leaves during late plant development was due to onset of grain filling as the carbohydrate pool in rice leaves were increasingly mobilized and exported to the developing grain. Rice leaves appear to store more sucrose than starch. The doubled-CO2 treatments consistently had greater SPS activity, which was associated with and helped maintain relatively higher sucrose
81




82
concentration in leaves. Increased SPS activity could have originated from increased SPS protein or from a kinetically more active form in high-C02 leaves.
Water deficit at panicle initiation (Figure 5.1) substantially decreased sucrose as well as starch concentration and also SPS activity. When water was witheld during panicle initiation, SPS activity, leaf sucrose and starch concentration declined concurrently from the onset of stress through the most stressed leaf phase. After water restoration, sucrose concentration and SPS activity rapidly recovered, but starch concentration slowly recovered. After plants regained turgor there was slower recovery in leaf starch concentration for up to 7 days.
Water deficit at anthesis caused considerable decrease in sucrose, starch, and SPS activity. Repeated stressed leaves were relatively higher in sucrose and starch concentration and SPS activity compared to water-stressed leaves at anthesis. Water withholding during anthesis phase decreased the plant's ability to recover from declining SPS activity, leaf sucrose and starch concentration was lower compared to that of plants exposed to water stress in the panicle initiation phase.
It is possible to conclude that under water stress the higher sucrose concentration over starch concentration was due to the increases in the ratio of newly synthesized sucrose to starch and to the degradation of starch, together with the reduction in sucrose export out of the leaves. The doubled-




83
80
0
40,
20
54o 64 74- 84 94 104 114 124
1804 350FLD
0- 700FLD
700DPI 40 "70P
Cl 54 64 74 84 94 104 114 124
'70
0.
E 50 T co u
%
20.
S1054a A a
O 54 64 74 84 94 104 114 124 Days After Planting (DAP)
Figure 5.1. Time course of sucrose and starch concentration and SPS activity under continuously-flooded and water-deficit at panicle initiation treatments.




84
100
so,-.'%o
80*
C%
60
C
8 0
i 2- 0
CO 54 64 74 84 94 104 114 124
9 100
o80 350DAN
1 -0- 700DAN
60 -** 350DBS
* .-- 700DBS 00 -W 70ODBS
40 '
20
S 54 64 74 84 94 104 114 124
70
a60 **
E 50 9-40 % % %,
- 40
D 54 64 74 84 94 104 114 124
Days After Planting (DAP)
Figure 5.2. Time course of sucrose and starch concentration and SPS activity under water deficit at anthesis and at both phase treatments.




85
CO2 concentration was able to delay the effect of water deficit for one day.
Relationships of SPS and carbohydrate can also be evaluated via C02-enrichment effects on photosynthesis and soluble protein. CO2 enrichment increased Pn which would increase triose production for sucrose and starch formation. However elevated CO2 also decreased soluble protein and presumably also Rubisco, which is a major constituent of soluble protein. The question is why is SPS activity increased despite a decrease in soluble protein. One
possibility is that increasing Pn increased hexose formation which led to selective mRNA transcription to enhance synthesis of SPS protein or of an SPS activase protein that causes increase in SPS activity. Another possibility is that
inorganic phosphate, Pt, in the cytosol will decrease under CO2-enrichment and higher triose-P production. Decreasing P1 levels causes dephosphorylation of SPS which results in increasing SPS activity. This hypothesis assumes that the SPS assay determines only the kinetically active form of SPS. Increasing SPS activity would increase sucrose production. Therefore, rice leaves partition more assimilate to sucrose rather than starch.
Relationships of SPS and carbohydrates under water deficit can likewise be evaluated in terms of drought effects on Pn and soluble protein. When the plants were subjected to drought stress by withholding water, the initial response of




86
the plant is stomatal closure. This response will inhibit C02 diffusion into leaves which will decrease photosynthetic rate and result in decreased carbohydrate formation and deactivation of SPS activity (Vassey et al., 1991). The cause of decreased SPS activity under mild water deficit could be the reverse of events under elevated C02, i.e., increased Pi availability may cause phosphorylation of SPS thus decreasing SPS activity or less hexose could lead to less transcription for synthesis of SPS protein. A third cause for decreased SPS activity is possible. Water deficit also causes
photoinhibition and increases protein turnover as well as increases proline and glycine betaine production. That was evident in this study from decreased soluble protein under water deficit. This would lead to decrease in SPS protein, which in turn would decrease SPS activity. Photoinhibition also broke down chlorophyll, thus resulting in decreased ATP and NADPH formation and also decreased CO2 assimilation.
Elevated CO2 promoted larger reduction in leaf stomatal conductance. As a result, the plant stands grown at elevated CO2 utilized the available soil moisture more slowly than those grown under ambient CO2. High-CO2 plants also decreased transpiration rate more compared to ambient-CO2 plants under water deficit. Therefore, plants grown in CO2-enriched environment were able to partly compensate for the effect of water stress and to extend the growing period.




CHAPTER 6
SUMMARY AND CONCLUSION
The global atmospheric CO2 concentration and regional rainfall intensities and frequencies are predicted to change during the next century. These anticipated changes in CO2 and water availability could have adverse ef fects on physiology of rice, based on previous elevated-CO2 studies on rice (Baker et al., 1988, 1996a, 1996b; Rowland-Bamford et al., 1990; Makino et al., 1994), bean (Vassey and Sharkey, 1989; Vassey et al., 1991) and spinach (Zrenner and Stitt, 1991). The objective of this study was to evaluate the effect of doubled CO2 and specific water deficits on rice photosynthesis, soluble
protein and chlorophyll concentration, carbohydrates, and SPS activity. This chapter briefly describes and summarizes the results of this study.
Photosynthesis, Soluble Protein and Chlorophyll
Elevated CO2 concentration increased leaf photosynthesis 40% throughout the season. water deficit initiated on the
same date decreased photosynthetic rate of ambient-CO2 leaves significantly earlier (about 1 day) than the rate of elevatedCO2 leaves. For example, at day 74 during the panicle initiation water deficit, reduction in photosynthetic rate due 87




88
to water deficit was 96% in low-CO2 leaves and 53% in high CO2 leaves during the panicle initiation phase.
Leaf soluble protein of elevated-CO2 leaves was significantly lower on most dates of the growing season. The greatest differences in leaf soluble protein between high-C2 and low-CO2 plants was 18%. Withholding water during panicle initiation decreased leaf soluble protein concentration by 62% in low-CO2 leaves and by 56% in doubled-CO2 leaves. Effects of water deficit during anthesis on leaf soluble protein were significant. Anthesis drought stress decreased soluble protein concentration by 62% in ambient-CO2 and 55% in elevated-CO2 plants.
The average increase in chlorophyll concentration for enriched-CO2 over ambient-CO2 plants was 13%. During water deficit, elevated-CO2 leaves had significantly higher chlorophyll concentration than low-CO2 leaves. Reduction in chlorophyll concentration during water deficit in the panicle initiation phase was 62% in ambient-CO2 leaves and 48% in elevated-CO2 leaves. During anthesis phase, water deficit decreased chlorophyll 73% in low-CO2 plants and 54% in high-CO2 plants. Rice plants exposed to two water stress cycles had decreased chlorophyll concentration of 76% in low-CO2 leaves and of 62% in high C02 leaves.
Carbohydrates
Elevated CO2 concentration significantly increased sucrose concentration throughout the season by 9%. Water




89
deficit during panicle initiation decreased sucrose concentration by 85% in ambient-C02 and 85% in elevated-CO2 leaves. Effects of water management treatment during anthesis significantly decreased concentration of sucrose by 69% in ambient-CO2 and 58% in doubled-CO2 leaves.
Leaf starch concentration was significantly increased by [C02] treatments from 58 through 74 DAP. Effects of [CO2] treatment on concentration of starch declined with plant maturity. During panicle initiation, water deficit decreased leaf starch concentration by 93 % in low-CO2 and 92% in highCO2 leaves. Water deficit during anthesis also significantly decreased starch concentration.
Leaf fructose concentration increased by 36% with doubling [CO2] from 54 through 127 DAP. Effects of [CO2] treatments on fructose concentration were significant. Water deficit during panicle initiation significantly decreased fructose concentration by 90% in low-CO2 and 84% in elevatedCO2 leaves. When water was withheld during the anthesis phase, fructose concentration decreased by 80% in low-CO2 leaves and 45% in high-CO2 leaves.
SPS Activity
Elevated [CO] increased SPS activity throughout the season by 6 to 16%. Water stress treatment during the panicle initiation phase significantly decreased SPS activity by 72% in ambient-CO2 and 58% in doubled-CO2 plants. When water was




Full Text

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THE EFFECT OF ELEVATED CO 2 AND WATER DEFICIT ON PHOTOSYNTHESIS AND PHOTOSYNTHATE PARTITIONING OF RICE LEAVES By WIDODO A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1996

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ACKNOWLEDGMENTS I wish to express my sincere appreciation to committee members Dr. George E. Bowes, Dr. Cu v. Vu, Dr. Leon H. Allen Jr., Dr. Raymond N. Gallaher and Dr. Kenneth J. Boote. The direction and support they have provided during my doctoral studies has been invaluable. I would like to give special thanks to my committee chairman and advisor, Dr. Kenneth J. Boote, for his numerous ideas, patience, and guidance throughout my research. I would like further to thank Drs. L.H. Allen Jr., K.J. Boote, and C.V. Vu for providing the oportunity to carry out the CO 2 enrichment study on rice and for providing all the necessary facilities for carbohydrates and enzyme analyses. I would like to extend my appreciation to Dr. J.T. Baker for conducting whole-canopy chamber experiments, and for his suggestions and help. Jean Marie Thomas, Joan Anderson and Pan Deyun deserve many thanks. They offered invaluable help and guidance for my laboratory and field studies. I thank my parents who taught me the values needed to complete this degree. I also thank my children, Okky and Intan, whose cheerfulness gave added incentive to complete my studies. The greatest thanks go to my wife Pratiwi ii

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Widiastuti, whose patience, hard work, and understanding allowed me to accomplish my degree. Finally, I would like to acknowledge the Higher Education Development Projects of Indonesian Government for providing scholarship and financial assistance under the USAID program. iii

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TABLE OF CONTENTS ACKNOWLEDGMENTS LIST OF TABLES LIST OF FIGURES ABSTRACT CHAPTERS 1 LITERATURE REVIEW Introduction ..... Effects of CO 2 on Photosynthetic Enzymes Effects CO 2 and Drought Stress on Soluble Protein and chlorophyll ...... Effects of CO 2 and Water Stress on Sucrose Synthesis Enzymes Effects of CO 2 and Water Stress on Photosynthesis .......... ii vi ix X 1 1 4 6 7 10 Effects of CO 2 and Water Stress on Non-Structural Carbohydrates . . . . 20 2 EFFECTS OF ELEVATED CO 2 CONCENTRATION AND WATER DEFICIT ON RICE LEAF PHOTOSYNTHESIS, CHLOROPHYLL AND SOLUBLE PROTEIN . . . 2 8 Introduction ..... Materials and Methods .. Results and Discussion 28 31 35 3 RICE LEAF NONSTRUCTURAL CARBOHYDRATES IN RESPONSE TO ENHANCED CO 2 AND WATER STRESS . 50 Introduction ..... Materials and Methods Results and Discussion 4 EFFECTS OF ELEVATED CO 2 CONCENTRATION AND WATER 50 53 55 STRESS ON SUCROSE PHOSPHATE SYNTHASE ACTIVITY 71 Introduction 71 iv

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Materials and Methods .. Results and Discussion ... 5 COORDINATION OF CARBON METABOLISM ACTIVITIES 6 SUMMARY AND CONCLUSIONS REFERENCE LIST BIOGRAPHICAL SKETCH V 74 75 81 87 91 107

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LIST OF TABLES Table 2.1 Times when water was withheld and restored for drought treatments for all chambers. Rice was planted on 15 July 1994 . . . 34 2.2 Leaf photosynthetic rate of rice plants grown at 350 and 700 L CO 2 L 1 and in four different water managements. Measurements were taken on 51, 55, and 60 day-old plants (before stress affected) and 72 day-old plants (early water deficit period) . . . . . 42 2.3 Leaf photosynthetic rate of rice plants grown at 350 and 700 L CO 2 L 1 and in four different water managements. Measurements were taken at noon eastern standard time from 74 (stressed), 75 (stressed), 82 and 84 (recovery) day-old plants. 43 2.4 Leaf photosynthetic rate of rice plants grown at 350 and 700 L CO 2 L 1 and in four different water managements. Measurements were taken on 89, 98 and 100 day-old plants ............. 44 2 5 Leaf photosynthetic rate of rice plants grown at 350 and 700 L CO 2 L 1 and in four different water managements. Measurements were taken from 109, 114 and 125 day-old plants .......... 45 2.6 Leaf soluble protein concentration of rice plants grown at 350 and 700 L CO 2 L 1 and in four different water managements. Measurements were taken from 54, 74, 82 and 92 day-old plants 46 2.7 Leaf soluble protein concentration of rice plants grown at 350 and 700 L CO 2 L 1 and in four different water managements. Measurements were taken from 100, 110 and 127 day-old plants ..... 47 2.8 Leaf chlorophyll concentration of rice plants grown at 350 and 700 L CO 2 L 1 and in four different water managements. Leaf samples were taken from 54, 74, 82 and 92 day-old plants .... 48 vi

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2.9 Leaf chlorophyll concentration of rice plants grown at 350 and 700 L CO 2 L 1 and in four different water managements. Measurements were taken from 100, 110 and 127 day-old plants .. 49 3.1 Leaf sucrose concentration of rice plants grown at 350 and 700 L CO 2 L 1 and in four different water managements. Leaf samples were taken at noon eastern standard time from 55, 58, 72 and 74 day-old plants . . . .. 62 3.2 Leaf sucrose concentration of rice plants grown at 350 and 700 L CO 2 L 1 and in four different water managements. Leaf samples were taken at noon eastern standard time from 75 (stressed), 82 and 89 (recovery) day-old plants . .. 63 3.3. Leaf sucrose concentration of rice plants grown at 350 and 700 L CO 2 L 1 and in four different water managements. Leaf samples were taken at noon eastern standard time from 100, 109 (deficit period) and 125 (recovery period) day-old plants ................... 64 3.4. Leaf starch concentration of rice plants grown at 350 and 700 L CO 2 L 1 and in four different water managements. Leaf samples were taken at noon eastern standard time from 55, 58, 72 and 74 day-old plants .............. 65 3.5 Leaf starch concentration of rice plants grown at 350 and 700 L CO 2 L 1 and in four different water managements. Leaf samples were taken at noon eastern standard time from 75 (stressed), 82 and 89 (recovery) day-old plants ... 66 3.6 Leaf starch concentration of rice plants grown at 350 and 700 L CO 2 L 1 and in four different water managements. Leaf samples were taken at noon eastern standard time from 100, 109 (stressed) and 125 (recovery) day-old plants .... 67 3.7. Leaf fructose concentration of rice plants grown at 350 and 700 L CO 2 L 1 and in four different water managements. Leaf samples were taken at noon eastern standard time from 55, 58, 72 and 74 day-old plants . . .. 68 3.8 Leaf fructose concentration of rice plants grown at 350 and 700 L CO 2 L 1 and in four different water managements. Leaf samples were taken at noon eastern standard time from 75 (stressed), vii

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82 and 89 (recovery) day-old plants 3.9 Leaf fructose concentration of rice plants grown at 350 and 700 L CO 2 L 1 and in four different water managements. Leaf samples were t aken at noon eastern standard time from 100, 109 69 (stressed) and 125 (recovery) day-old plants .... 70 4.1 Sucrose phosphate synthase activities of rice plants grown at 350 and 700 L CO 2 L 1 and in four different water managements. Plant samples were taken at noon eastern standard time from 54, 74 (stressed), 82 and 92 (recovery) day-old plants . . . . . 79 4.2 Sucrose phosphate synthase activities of rice plants grown at 350 and 700 L CO 2 L 1 and in four different water managements. Plant samples were taken at noon eastern standard time from 100, llO(stressed) and 127 (recovery) day-old plants ......... ; ......... 80 viii

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LIST OF FIGURES Figure 5.1. Time course of sucrose and starch concentration and SPS activity under continuously-flooded and water deficit at panicle initiation treatments . 83 5.2. Time course of sucrose and starch concentration and SPS activity under water deficit at anthesis and at both phase treatments . . . 84 ix

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy THE EFFECT OF ELEVATED CO 2 AND WATER DEFICIT ON PHOTOSYNTHESIS AND PHOTOSYNTHATE PARTITIONING OF RICE LEAVES By Widodo December 1996 Chairperson: Kenneth J. Boote Major Department: Agronomy The carbon dioxide concentration of the earth's atmosphere and frequency and intensity of rainfall are predicted to change in future years. The resulting changes could have considerable impact on crop production. The objective of this study was to evaluate responses of rice leaves to CO 2 concentration and water management for rice grown in controlled environmental chambers. Leaves of rice plants responded differently to CO 2 concentration and various water managements. High-CO 2 concentration significantly increased leaf photosynthetic rate during the study. On several dates, leaf soluble protein responded negatively by decreasing under increased CO 2 concentration. Nevertheless, leaf chlorophyll concentration of leaves of high-CO 2 treatments was significantly higher compared to that of low CO2 treatments. High-CO 2 treatments significantly increased X

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leaf sucrose, starch and fructose concentration by 0. 0 to 25.8%, Oto 40.9% and 12.2 to 64.0%, respectively. High-CO 2 concentration had significant enhancing effects on sucrose and starch accumulation during early reproductive phases, but not during later reproductive phases. The difference in leaf starch, sucrose, and fructose concentration between leaves grown at elevated and current CO 2 diminished with plant maturity. High-CO 2 concentration also increased SPS activity throughout the season. Water stress treatment significantly affected a number of variables during panicle initiation and anthesis stages. Water stress caused major reductions in leaf photosynthetic rate, leaf chlorophyll, and soluble protein, and water deficit periods also caused major decreases in leaf sucrose, starch and fructose concentration, and also in SPS activity. Water stress had more profound effects on leaves of plants grown in low-CO 2 concentration. High-CO 2 plants were able to maintain leaf photosynthesis longer into the water deficit period and had smaller reductions in chlorophyll and fructose concentration compared to ambient CO 2 plants. xi

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CHAPTER 1 LITERATURE REVIEW Introduction The amount of carbon dioxide (CO 2 ) in the earth's atmosphere has risen over the last century because of human activities that release CO 2 from terrestrial reservoirs (Roeckner, 1992). These activities include energy production and use, manufacturing and agricultural processes, land use changes, and waste disposal (Subak et al., 1993). Over the past four decades, observations of atmospheric concentration ([CO 2 ]) at Mauna Loa, Hawaii, and at the South Pole show an approximate proportionality between the rising atmospheric concentrations and industrial CO 2 emissions (Keeling et al 1995). During the past 200 years, the atmospheric [CO 2 ] has increased from 280 ppmv to 360 ppmv. Atmospheric [CO 2 ] is expected to reach twice the preindustrial level by the middle of the next century (Keeling et al., 1995). Ice core analyses of polar ice has revealed a high correlation between climatic change and variations in the atmospheric concentrations of greenhouse gases (CO 2 and methane) over the last 160,000 years (Roeckner, 1992). When greenhouse gas concentration increases, atmospheric moisture variability is substantially larger over areas that 1

PAGE 13

2 experience the greatest surface warming (Liang et al., 1995). Results from a coupled biosphere-atmosphere model (SiB2-GCM) indicate that, for doubled CO 2 conditions, evapotranspiration will decrease and air temperature will increase over the tropical continents, amplifying the changes resulting from atmospheric radiative effects (Sellers et al., 1996). Moreover, the global mean surface temperature will rise between 1. 5 c and 4. 5 c (Houghton et al. 19 9 5) In addition, under double CO 2 condition, variability of daily precipitation will increase. There will be some areas with increases and decreases of frequency and intensity of precipitation (Mearns et al., 1995). The resulting precipitation shifts could have considerable agricultural impact especially in regions that become drier (Wigley et al., 1980). Since soil water availability currently limits crop growth more than all other environmental factors combined (Boyer, 1982), it is important to investigate the effects and possible interactions of water regimes with [CO 2 ] upon major food crops such as rice. Rice (Oryza sativa L.) provides over half the diet of 1.6 billion people and is the only grain crop used almost exclusively for human food consumption. World rice production is second after wheat production. Rice is grown over a wide range of latitudes from roughly 53 N to 40 s and to elevations of more than 2500 m (Mooreman and van Breeman, 1978). Of the total of 140 to 145 million hectares of land

PAGE 14

3 area planted to rice annually (IRRI, 1982), nearly 12% of the world's rice area is devoted to upland rice culture. Upland rice yields are lower than lowland rice, mainly due to complete dependence on rainfall for moisture (De Datta and Beachell, 1972). Moreover, about half of the world's rice land area depends on rainfall and is often subjected to drought stress (IRRI, 1982). Several researchers have conducted studies of the effects of rising [CO 2 ] on rice (Yoshida, 1976; Baker et al., 1990; Baker et al., 1992; Ziska and Teramura, 1992a, 1992b; Ziska et al. 199 6) In response to superambient CO 2 rice photosynthesis, biomass, grain yield, and leaf carbohydrates increased up to about 500 bar CO 2 (Yoshida, 1976); but above this, gains were minimal (Baker et al., 1990; Baker et al., 1992). The photosynthetic and growth response to elevated CO 2 may be highly dependent on the supply of N (Ziska et al., 1996). The availability of water is one of the most important factors determining vegetation diversity and plant productivity (Rochefort and Woodward, 1992). The effects of water deficits on plant performance and growth are mediated through decreases in stomatal conductance and photosynthesis and depend on the severity and duration of the drought period, the presence of further environmental constraints, and species inherent characteristics (Chaves and Pereira, 1992). Water stress can have profound metabolic effects in plants,

PAGE 15

4 resulting not only in impaired gas exchange but also in profound alteration of physiological processes, such as cell growth, wall synthesis, nitrogen and chlorophyll metabolism, and the levels of growth substances (Hsiao, 1973). Rice is most sensitive to water stress around the flowering stage (Yoshida, 1978). Moisture stress at anthesis results in the failure of the panicle to exert fully from the flag leaf sheath. Reduced panicle exertion was shown to be regulated by the plant water status. A direct cause and effect relationship was also noted between panicle exertion and spikelet fertility (O'Toole and Namuco, 1983). In the rice plant, 60-90% of the total carbon in panicles is derived from photosynthesis after heading, and the flag leaf is the organ that contributes the most to grain filling (Yoshida, 1981). Under water stress (Boyer, 1976; Kreidemann and Downton, 1981; Sinha et al. 19 82; Yoshida, 1978) and (Clifford et al. 1993; Jones, 1993; Yoshida, 1976) CO 2 enrichment induces changes in crop growth, metabolites and enzymes associated with assimilatory and degradative reactions in plants. Effect of CO 2 on Photosynthetic Enzymes The enzyme responsible for initiating C 3 photosynthesis, rubisco, has been the focus of much attention regarding the regulation of the rate of carbon entering the photosynthetic pathway. Rubisco, ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco), catalyses two different

PAGE 16

5 reactions in the chloroplast stroma (Campbell et. al., 1988). This enzyme acts as carboxylase with the substrates RuBP and CO 2 and as an oxygenase with the substrates RuBP and 0 2 The former reaction results in CO 2 uptake of photosynthesis whereas the latter results in mitochondrial CO 2 evolution called photorespiration. Thus the net rate of assimilation of CO 2 in a leaf depends on the rates of RuBP carboxylation and RuBP oxygenation. The ratio of carboxylation to oxygenation is determined by the concentration of the substrates CO 2 and 0 2 and by the catalytic properties of the enzyme with respect to these substrates (Brooks and Farquhar, 1985). The dependence of Rubisco activity (carbamylation) on PFD can be altered by CO 2 0 2 and temperature. For example, under atmospheric conditions (21% 0 2 and 350 ppm CO 2 ) and normal temperature, rubisco activity generally varies with PFD by only two to three fold. However, at 2% 0 2 and below, a reduction of greater than 90% of maximal Rubisco activity can be observed at very low PFD. In some cases, especially under high CO 2 and low temperatures (14 C), the changes in Rubisco activity appear to be correlated with a reduced ATP/ADP ratio associated with a reduction in photosynthesis by a "feedback effect" (Portis, 1992). In intact C 3 plants, increasing temperature causes an increase in both the CO 2 compensation point and the inhibition of net photosynthesis by 0 2 This suggests that the rate of photorespiration relative to the

PAGE 17

6 rate of photosynthesis increases with temperature (Machler and Nosberger, 1980). Effect of CO 2 and Drought Stress on Soluble Protein and Chlorophyll Schwanz et al. (1996) reported that antioxidants, protective enzymes, soluble protein, and pigments showed considerable fluctuations in a two year experiment on oak (Quercus robur) and pine (Pinus pinaster) seedlings in response to enhanced [CO 2 ] and drought stress. When the seedlings were subjected to drought stress by withholding water, the activities of antioxidative enzymes decreased in leaves of pine and oak grown at ambient [CO 2 ] and increased in plants grown at elevated [CO 2 ]. The authors suggested that growth in elevated CO 2 might reduce oxidative stress to which leaf tissues are normally exposed and enhance metabolic flexibility to encounter increased stress by increases in antioxidative capacity. Ficus opuntia (Jacob et al., 1995) grown at elevated [CO 2 ] also contained less soluble protein ( 39 -52%) The rubisco content was 4 3 to 58% of soluble protein. Leaf chlorophyll content per unit area or dry mass was significantly lower in elevated CO 2 -grown plants and increased significantly with increasing nutrient availability (Radoglou and Jarvis, 1992). Hunt et al. (1996) found that after two growing season, plant tissue N concentrations of C 3 and C 4 grasses were lower under elevated CO 2

PAGE 18

7 Effects of CO 2 and Water Stress on Sucrose Synthesis Enzymes When stomata close in response to water stress, CO 2 concentration inside the leaf goes down, ultimately leading to deactivation of sucrose phosphate synthase (SPS) activity. This decrease in SPS activity shows up in measurements of gas exchange under water stress even though it may be a consequence of low CO 2 (Vassey et al 1991). When water stress occurs for several days, the reduction in SPS capacity is no longer reversed by a 20 min incubation in high CO 2 but requires 5 h for recovery. These results indicate that the activity of SPS can be influenced by the CO 2 concentration surrounding the plant (Vassey et al., 1991). Sucrose Phosphate Synthase Sucrose Phosphate Synthase (SPS) plays an important role as the limiting factor of partitioning of carbon sources. Therefore, it seems that enhancement of this activity may promote the ability of the source function (Stitt and Quick, 1989). Using isolated maize SPS cDNA which is over-expressed using the promoter of the Rubisco small subunit gene in transgenic tomato, SPS activity was increased six fold in leaves, resulting in doubling of sucrose content but significant reduction of starch in these leaves (Worrell et al. 1991).

PAGE 19

8 SPS is allosterically activated by the presence of glucose-6-phosphate, but inhibited by increase of inorganic phosphate, P i (Stitt and Quick, 1989). In addition, there is a covalent modification by phosphorylation/dephosphorylation, which regulates its enzyme activity (Walker and Huber, 1989; Huber and Huber, 1992). This modification occurs in response to light and darkness, showing circadian rhythms. In leaves, illumination induces dephosphorylation which leads to the enzyme being activated, and darkness results in inactivation because of phosphorylation (Kalt-Torres et al., 1986; Stitt et al., 1988; Huber and Huber, 1991). Sakamoto et al. (1995) suggested that function and promoter of rice SPS gene is weak and no typical promoter sequences were found upstream of the coding region. The deduced amino acid sequence of the rice SPS showed a high degree of homology to the known ones. Expression of this gene was detected only in the leaves, suggesting that this gene is specifically expressed in the source organs. Level of expression was extremely low, reflecting weakness of its promoter activity. After panicle removal at anthesis, Nakano et al. (1995) found that activity of SPS remained relatively high during leaf senescence and decreased more slowly than that in the control plants. Vassey et al. (1991) reported that SPS activity was low in plants held in low CO 2 for 1 h. The low-CO 2 inhibition of SPS activity could be reversed by incubation of

PAGE 20

9 the leaf tissue in high CO 2 concentration and high light for 20 min. The activity of soybean SPS was unchanged by long-term CO 2 -enrichment (Huber et al., 1984), but it increased in rice (Hussain et al., 1992). Vassey et al. (1991) concluded that the CO 2 effect on SPS activity was mediated by the effect of CO 2 on the rate of photosynthesis. Micallef et al. (1995) reported that tomato leaves of SPS-transformed lines were significantly greater (up to 12 times) in limit i ng and maximum SPS activities. Partitioning of carbon into sucrose increased 50% for the SPS transformants. Intact leaves of the control plants exhibited CO 2 insensitivity of photosynthesis at high CO 2 levels, whereas the SPS transformants did not exhibit CO 2 -insensitivity. Growth at 65 Pa CO 2 resulted in reduced photosynthetic capacity for control plants but not for SPS transformed plants. When grown at 65 Pa CO 2 SPS-transformed plants had a 20% greater photosynthetic rate than controls when measured at 65 Pa CO 2 and 35% greater rate when measured at 105 Pa CO 2 Furthermore, transgenic tomato plants expressing high levels of maize SPS have been concluded to support the postulate that SPS activity can influence the partitioning of carbon between starch and sucrose (Huber and Huber, 1992). In addition, SPS of maize leaves was found in both the mesophyll and bundle sheath cells (Huber et al., 1987). High [CO 2 ] increased leaf blade elongation rate (LER) of expanding blades and in vivo activity (V 1 tmtttng ) SPS activity of

PAGE 21

10 expanded blades during the early vegetative phase of rice (21 dafter planting [DAP]), when tillers were becoming strong carbohydrate sinks. There was a distinct diurnal pattern in LER, SPS activity, and concentration of soluble sugar, with an increase in the early part of the light period and a decrease later in the light period. The higher SPS activity at elevated CO 2 at 21 DAP was caused by an increase in the activation state of the enzyme rather than an increase in Vmax. By the midtillering stage (42 DAP) CO 2 enrichment enhanced SPS activities of source blades (Seneweera et al., 1995). Effects of CO 2 and Water Stress on Photosynthesis Effects of CO 2 on Photosynthesis Many studies have shown that several growth parameters, including leaf area and dry weight, are enhanced during long term exposure to high CO 2 (Wong, 1979). However, after the initial stimulation of net photosynthetic rate per unit of leaf area by CO 2 enhancement may decrease during the subsequent exposure to high CO 2 and a subsequent suppression of photosynthesis may occur (Yelle et al., 1989). The direct effects of [CO 2 ] enrichment on rice (Oryza sativa, L.), soybean (Glycine max, L.) and citrus (various species) was always an increase in photosynthetic rate (Baker and Allen, 1993). Furthermore, photosynthetic rate of rice measured at ambient [CO 2 ] decreased with increasing long-term [CO 2 ] growth

PAGE 22

11 treatment due to a corresponding decline in RuBP carboxylase content and activity. Prolonged exposure to high CO 2 leads to changes in biochemical, physiological or morphological factors which may remove or offset the initial stimulation of photosynthesis (DeLucia et al., 1985). Results of experiments with CO 2 upon photosynthesis vary with the species investigated. Nevertheless, by manipulating the CO 2 c8ncentration, CO 2 can be used to probe the responses of various photosynthetic parameters and to aid in determining their role in regulation of photosynthesis (Kramer, 1981). However, it has been established that acclimation to elevated CO 2 alone may lead to a subsequent decline in photosynthetic rate, for which a variety of causes have been invoked, some related to aspect of leaf structure and functioning (Rogers et al., 1983; Sage et al., 1988). However, Idso and Kimball (1991) reported that after a full 3-year period, CO 2 -enriched sour orange trees grown under irrigation and high fertility in the desert environment at Phoenix, Arizona, USA, had consistently sequestered approximately 2. 8 times more carbon than the control trees. The authors suggested that these plants may not experience the downward regulation of photosynthetic capacity under field conditions i n the natural environment, compared to long term CO 2 enrichment plants grown in pots On the other hand, Allen and Amthor (1985) pointed out that both the CO 2 -enriched and the nonenriched sour orange

PAGE 23

12 trees exhibited midday (and afternoon) depression of photosynthesis under the hot summertime conditions of Phoenix, Arizona. Furthermore, the depression was much more severe in the nonenriched trees than the enriched trees, with leaf photosynthetic rates for the nonenriched trees being only about 1 mol CO 2 m 2 s 1 for the last half of the day. Apparently, elevated CO 2 provided protection against midday depression of photosynthesis by some unknown mechanism not related to downregulation. Ziska and Teramura (1992b) revealed that two rice cul ti vars (IR-36 and Fujiyama-5) increased 50% in photosynthetic rate when exposed to enhanced [CO 2 ] (660 bar) and photosynthetic enhancement was still evident after 3 months of exposure to a high CO 2 environment. However, in plants exposed to simultaneous increases in CO 2 and ultraviolet-B (UV-B) radiation, CO 2 enhancement effects on respiration, photosynthesis, and biomass were eliminated in IR-36 and significantly reduced in Fujiyama-5 (Ziska and Teramura, 1992a). Balaguer et al. (1995) concluded that doubling the atmospheric concentration of CO 2 enhanced the rate of net CO 2 assimilation by 47%. Zhang and Nobel (1996) studied a C 3 desert shrub (Encelia farinosa) and found an increase in assimilation rate by 46% in the early morning, 26% at midday, and 15% in the late afternoon. Balaguer et al. (1995) also found that doubling [CO 2 ] reduced the proportion of fixed

PAGE 24

13 carbon retained in the leaf blade, increasing the rate of export. The favorable carbon balance of CO 2 enriched leaves was further enhanced by a decrease in the cost of maintenance respiration. Jacob et al. (1995) reported that Scirpus olneyi grown at elevated CO 2 had a significantly higher (30-59%) net CO 2 assimilation rate than plants grown at ambient CO 2 when measurements were performed on excised stems at the respective growth [CO 2 ]. However, when measurements were made at normal ambient [CO 2 ], net CO 2 assimilation rate was smaller (45-53%) in plants grown at elevated [CO 2 ] than in those grown at ambient [CO 2 ]. Changes in carboxylation efficiency and in situ carboxylase activity were caused by a decreased rubisco concentration (30-58%) in ~lants grown at elevated [CO 2 ]. Van Oosten and Besford (1995) revealed that thylakoid proteins (photosystem I core protein, D 1 and D 2 of the photosystem II core complex, cytochrome f) were all decreased by elevated CO 2 after 31 days exposure on the fully mature leaves of tomato plants, whereas the large and small subunits of rubisco and Rubisco activase proteins had already declined after 22 d exposure. Ziska et al. (1996) reported that rice photosynthesis was initially stimulated at the leaf and canopy level with elevated CO 2 regardless of supplemental N supply, but with time the photosynthetic response became highly dependent on

PAGE 25

14 the level of supplemental N, increasing proportionally as N availability increased. Baxter et al. (1995) reported that photosynthetic capacity of Paa alpina was reduced by growth at 680 rnol mo1 1 CO 2 after 105 d, and that of Fectusa vivipara L. was reduced at 65 d and 189 dafter CO 2 enrichment began, suggesting down regulation or acclimation. In F. vivipara the relationship between leaf photosynthetic capacity and leaf carbohydrate concentration was such that there was a strong positive correlation between photosynthetic capacity and total leaf N concentration (expressed on a per unit structural dry weight basis), and total nitrogen concentration of successive mature leaves decreased with time. Atmospheric CO 2 enrichment is typically associated with increased rates of leaf photosynthesis and total plant dry matter accumulation. However, increased photosynthetic rates per unit leaf area may not persist for long periods at high atmospheric CO 2 concentration (Clough et al., 1980). Ho (1977) reported increased rates of photosynthesis and carbon transport in leaves of tomato plants grown under CO 2 enrichment, compared to plants grown at ambient CO 2 Barnes et al. (1995) reported that the increase in photosynthesis of Norway spruce (Picea abies [L.] Karst) induced by CO 2 enrichment was associated with increased foliar concentrations of glucose, fructose and starch (but no change in sucrose) in the new growth.

PAGE 26

15 Maximum carboxylation rates per unit leaf area (V cm ax) were lower in cotton leaves grown at two elevated CO 2 concentrations, compared with ambient CO 2 concentration, under all phosphorus and pot size treatments, indicating that acclimation of photosynthesis had occurred (Barrett and Gifford, 1995). The degree of photosynthetic acclimation to elevated CO 2 was not related to the degree by which whole plant carbon gain was stimulated by elevated [CO 2 ] at the different P supplies, or to the degree by which allocation to root and shoots was altered by pot size. Thus there was no simple relationship between photosynthetic and growth acclimation by cotton to elevated CO 2 At ambient CO 2 the maximum carboxylation rate increased linearly with an increase in leaf P per unit area (mg P m 1 ), but rates were lower at elevated CO 2 for a given P content m 2 Vcmax also increased linearly with an increase in leaf P concentration (mg P g 1 structural dry weight). However, values of Vcmax were similar for plants grown at ambient and elevated CO 2 for a given P concentration. Acclimation of photosynthesis at elevated CO 2 was associated with an increase in leaf starch determined 5 h into the light period. However, increased starch concentration with an increase in P supply was not associated with any decline in Vcmax (Barrett and Gifford, 1995).

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16 Effect of Water Stress on Photosynthesis One of the major injurious responses to water deficits is nonstomatal inhibition of photosynthesis, that is, inhibition of photosynthesis that cannot simply be ascribed to stomatal closure {Hsiao 197 3) Inhibition of leaf photosynthesis may result from a decrease in the conductance of CO 2 from the atmosphere to the chloroplasts, such as occurs when stomata close; from detrimental effect on the photosynthetic mechanism {mesophyll activity) itself; or from a combination of the two {Schulze, 1986). Leaf rolling is a common response by grasses to a water stress. This reduces water used, but also inhibits CO 2 assimilation per plant {Schulze, 1986). Boyer (1976) indicated that plant sensitivity to drought varies with the stage of growth. When leaves begin to desiccate, photosynthesis is inhibited and can be affected enough so that net carbon dioxide fixation ceases completely. Moreover, Yoshida (1978) reported that water stress affects both stomatal and nonstoma t al components of photosynthesis of rice. Initial photosynthet i c reduction is due to a decrease in the conductance of CO 2 t h rough stomata arising from plant water deficit. Furthermore, water stress reduces net photosynthate availability by reducing leaf area. This is followed by a decrease in the activities of enzymes such as RuBPCase and in the photochemical activity of the chloroplast (Sinha et al., 1982) Several investigators have demonstrated

PAGE 28

17 changes in chloroplast activity when chloroplasts are isolated from desiccated leaves. These changes involve a decrease in electron transport and photophosphorylation, and there are reports that CO 2 fixation by isolated chloroplasts is reduced (Boyer, 1976) At minimal leaf water potential, sorghum ( Sorghum bicolor L.) net photosynthesis was completely inhibited, with the stomata being closed (Contour-Ansel et al., 1996). After rewatering, plants showed recovery in photosynthesis but never reached the initial values. Water stress had a striking effect, both on net photosynthesis by regulation of stomatal aperture and Pyruvate P i Dikinase (PPDK) and Phosphoenol Pyruvate Carboxylase (PEPC ) activities. Therefore, the level of PPDK and PEPC activitie s may contribute to the limitation of photosynthetic CO 2 fixa t ion. Clifford et al. (1995) found that CO 2 exerted significant effects on groundnut stomatal frequency only in irrigated groundnut plants. The effe c ts of drought on leaf development outweighed the smaller effects of [CO 2 ], although reductions in stomatal frequency indu c ed by elevated atmospheric CO 2 were still observed. Elevated atmospheric CO 2 promoted larger reductions in leaf conduc t ance than the changes in stomatal frequency, indicating partial stomatal closure. As a result, the plant stands grown at elevated CO 2 utilized the available soil moisture more slowly than those grown under ambient CO 2 thereby possibly extending the growing period.

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18 Samarakoon and Gifford (1996a), studying cotton (Gossypium hirsutum cv. Sicala 34), found high-CO 2 plants decreased transpiration rate by 60% compared to that of lowCO 2 plants under water deficit. In addition, in wet soil maize transpiration rate was reduced on average by 29% at high CO 2 but neit he r tu tal dry matter nor plant height were significan t 1 y affected by CO 2 level (Samarakoon and Gifford, 1996b). In s oil that was drying from field capacity, plants in high CO 2 used about 30% less water than those in ambient CO 2 Kameli and Losel (1996) reported that inhibition of growth of wheat (Triticum c 1 rum L.) was only apparent when the water content of the plan t s tarted to decline. Dry weight of wheat continued to in c rea s during water stress. This cause d a sharp rise in sugar c ont ( ~ t, accounting for 20% of the gain in dry matter between days 27 and 31. Following re-watering, stressed plants increased leaf length and leaf area, and leaves regained turgidity after wilting. Growth inhibition coincided with a conside r able increase in sugar content. Photosynthesis rather than reserve starch was proposed to be the major source of sugar accumulated under water stress in durum wheat. Tomlinson and Anders : m (1995) reported that seedling biomass of red oak (Quercus rubra L.) increased with increasing CO 2 and decreased with water stress. Water stress shifted relative biomass distribution from stems to roots,

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19 whereas CO 2 did not alter distribution among leaves, stem, or roots. Photosynthetic rate increased with increasing CO 2 and decreased with water stress. Stomatal conductance decreased with both water stress and elevated CO 2 Photosynthetic water use efficiency was greater at elevated growth CO 2 but largely unaffected by water stress. Delgado et al. (1992) observed different patterns of photosynthesis in stressed and non-stressed plants of Nicotiana tabacum L. for which water stress reduced total net carbon fixation by 45\. Moreover, the decrease in dry mass production under water stress was related to a decrease in total leaf area per plant and decrease in cell number per unit leaf area. Jones (1993) reported that under irrigated conditions net photosynthesis of groundnut (Arachis hypogaea) leaves increased by approximately 40% when CO 2 was increased from 350 to 700 ppmv. Droughted plots in both CO 2 regimes exhibited negligible net photosynthesis after 85 DAS In addition, Clifford et al. (1993) observed that with groundnut grown in well-irrigated conditions, elevated CO 2 increased dry matter accumulation 15\ and pod yields 30\ (from 2.8 to 3.7 t ha 1 ). Under drought condition, elevated CO; increased groundnut dry matter production by 112% and yield by 468\ (from 0.22 to 1.25 t ha 1 ) as compared to plants grown at 350 ppmv CO 2

PAGE 31

Effects of CO 2 and Water Stress on Non-Structural Carbohydrates 20 Growth under long-term CO 2 enhancement can lead to carbohydrate accumulation (Allen et al., 1988; Baker et al., 1992; Rowland Bamford et al., 1990; Wong, 1990). This may be because the photosynthetic rate exceeds the sink capacity to utilize the photosynthate for growth. An apparent correlation between starch accumulation and suppression of photosynthesis has been often reported (Sasek et al., 1985). Although extreme enlargement of starch grains may lead to physical damage of the chloroplast (DeLucia et al., 1985) and also hinder CO 2 diffusion in the chloroplast, there is no evidence that starch accumulation directly inhibits photosynthesis. Another explanation is that starch accumulation in the chloroplast may occur when photosynthesis is suppressed by decreased capacity of orthophosphate (Pi) regeneration during starch and sucrose synthesis. Water-stressed leaves have decreased rates of starch synthesis and increased synthesis of sugars (Morgan, 1984). The author sugested that it is possible that changes in enzyme activities of the pathway leading to starch synthesis play a substantial role in altering partitioning during stress. When attached, translocating bean leaves were water stressed and partitioning was assayed by 10-min pulse labelling with 14 CO 2 starch accumulation was decreased by more than 75% (Vassey and Sharkey, 1989) In contrast, the

PAGE 32

21 accumulation of 14 C into the neutral fraction, which includes sucrose, was not significantly affected. Only a small fraction of [ 14 C]sucrose would be exported via phloem during the 10-min pulse duration in this study. Vassey and Sharkey (1989) showed that the ionic fraction, which includes organic acids and amino acids, increased from 4% (control) to 42% (stressed) of the recovered 1 4 C. Rhodes (1987) concluded that stressed leaves are known to synthesize praline and glycine betaine. Sucrose synthesis Sucrose synthesis is inhibited when sucrose accumulates in the leaf (Foyer, 1990), and this feedback inhibition seems to stimulate starch synthesis. Since feedback inhibition of sucrose synthesis leads to P 1 -regeneration limited photosynthesis in the short-term regulation, it is possible that starch accumulation may be an effective indicator of Pi regeneration limited photosynthesis. However, starch synthesis in the chloroplast also frees Pi, and actually starch accumulation can occur without suppression of photosynthesis (Stitt, 1991). In the flag leaf of wheat ( Tri ti cum aes ti vum) (Nie et al., 1995), soluble carbohydrate concentrations declined markedly with the onset of grain filling. However, carbohydrates that were stored in vegetative plant parts before heading made a smaller contribution to grain dry weight

PAGE 33

22 at [COJ below 330 mol mol 1 than for treatments at above ambient [CO 2 ] and increasing [CO 7 ] had no effect on the carbohydrate concentration in the grain at maturity (Rowland Bamford et al., 1990). Plants of Scirpus olneyi grown at elevated [CO 2 ] contained more nonstructural carbohydrates ( 25 53%) than those grown at ambient [CO 2 ] (Jacob et al., 1995). Plants grown at elevated [CO 2 ] appear to have sufficient sink capacity to utilize the additional carbohydrates formed during photosynthesis. Wang and Nobel (1995) reported that doubling the [CO 2 ] led to approx. 5% more sucrose, 560% more mannose and 17% less amino acids in the phloem exudate and also significantly increased mannose, starch and glucomannan in the chlorenchyma of Opuntia ficus indica. Moreover, Sicher et al. (1995) revealed that leaf starch and sucrose levels were greater in soybean plants grown at 70 than at 35 Pa CO 2 Partitioning into Starch and Sucrose Sucrose and starch are the principal end products of photosynthesis in most plants, and sucrose is the principal carbohydrate translocated from source to sink tissues (Stitt et al., 1987). During the day leaves accumulate sucrose and starch as well as exporting sucrose to the rest of the plant. At night the sucrose and starch stored during the day are mobilized to maintain export of sucrose to sink tissues and to support respiration in the leaf (Servaites et al. 1989). The

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23 types of carbohydrate stored in the leaves varies between species. In many species (e.g. soybean, potato and tomato) starch is the major leaf storage carbohydrate, while other species store either sucrose and starch (e.g. spinach) or mainly sucrose (e.g. wheat and barley) (Stitt et al., 1987). Lunn and Hatch (1995) found that ratio of primary partitioning into sucrose and starch varied from about 0.5 in those species that accumulated mostly starch in the leaves (Amaranthus edulis L., A triplex spongiosa F. Muell. and Flaveria trinervia (Spreng.) C. Mohr) to about 8 in Eleusine indica (L.) Gaertn., which accumulated mostly sucrose. Generally there was a reasonable link between the primary partitioning of photosynthate and the type of carbohydrate stored in the leaf during the day. It is also generally recognized that the starch content of Trifolium subteraneum leaves increases with increasing CO 2 concentration (Cave et al., 1981), while Zhang and Nobel (1996) reported that sucrose and starch contents of Encelia farinosa, a common c 1 desert shrub, increased during the day proportionally more than under the ambient CO 2 Galtier et al. (1995) reported that the rate of sucrose synthesis was increased relative to that of starch and sucrose/starch ratios were higher throughout the photoperiod in the leaves of all tomato plants expressing high SPS activity.

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24 Neutral sugars (predominantly sucrose) of wheat (Triticum aestivum) were found to be the most abundant form of carbohydrate accumulated by leaves during the day, but significant quantities of starch and high degree of polymerization (d p.) fructans were also present. Elevated CO 2 was found to have marked effects on diel patterns of export, storage and respiration, while the proportion of fixed carbon allocated to each of these processes were broadly similar (Balaguer et al., 1995). Combining high CO 2 and different levels of nitrogen (N) and phosphorus nutrition treatments on pea (Pisum sativum), Riviere-Rolland et al. (1996) found that phosphoenolpyruvate (PEP) carboxylase decreased, and chloroplast phosphate (P) translocator increased, in high CO ,. In contrast to Rubisco, down-regulation of PEP carboxylase was alleviated by low N and enhanced by low P. The increase in the Ptrans locator was little affected by N but was accentuated by low P. The increase in the P-translocator is considered to be one way of alleviating low P condition in the chloroplast and thus re balancing carbon partitioning between starch and soluble carbohydrates and amino acids. Riviere Rolland et al. (1996) proposed that acclimation of PEP carboxylase and translocator reflects adaptation to metabolic perturbations caused by high CO 2 There are conflicting reports as to the extent to which the extra carbon fixed (as a result of CO 2 enrichment) is used

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25 for export compared to storage (as starch) in leaves. Ho (1977) reported that enriched plants have a higher efficiency of carbon transport when transferred to, or grown at, elevated CO 2 In contrast, Finn and Brun (1982) suggested that the majority of the additional reduced carbon provided by CO 2 enrichment of soybean plants, was stored as leaf starch and was not available for transport to distant sink (roots and nodules). Starch and sucrose are the principal end-products of photosynthesis (Silvius et al., 1979 and Preiss, 1982). It has been postulated that the rate of sucrose formation indirectly controls the rate of starch formation (Silvius et al., 1979). Starch is deposited during the day exclusively in the chloroplast (Preiss, 1982). Patel and Mahapatra (1996) reported that sucrose was the major trans locatable sugar in the organs of fertile rice florets of both top and basal spikelets, and poor grain filling of the latter was not caused by deficiency of sucrose, but due to inhibition of sucrose supply of the external protective organs. Based on 1 4 C0 2 vulgaris, Sharkey et pulse-chase al. (1985) experiment reported on Phaseolus that sucrose formation was linearly related to assimilation rate (slope=0.35), while starch formation increased linearly with assimilation rate (slope=0.56) but did not occur if the assimilation rate was below 4 mol m 2 s 1 They indicated that the pathways for starch and sucrose synthesis are both

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26 controlled by the rate of net co assimilation, with sucrose the preferred product at very low assimilation rates. Nie et al. (1995) found that differences in soluble carbohydrate concentration between wheat leaves grown at elevated arid current ambient CO concentration diminished with crop development, while in rice leaf blades, the priority between the partitioning of carbon into storage carbohydrates or into export changed with developmental stage and [CO 2 ] (Rowland-Bamford et al., 1990). Moreover, during vegetative growth of rice, leaf sucrose and -starch concentrations increased with increasing [CO 2 ] but tended to level off above 500 mol mol 1 CO 2 The ratio of starch to sucrose concentration was positively correlated with the [CO 2 ]. At maturity, increasing [CO 2 ] resulted in an increase in total non-structural carbohydrate concentration in leaf blades, leaf sheaths and culms of rice (Rowland-Bamford et al., 1990). In C 3 plants, elevated atmospheric CO 2 concentrations can partially compensate for the negative effects of drought by increasing water-use efficiency and by sustaining larger net CO 2 assimilation rates at reduced stomatal conductance in leaves of stressed plants (Chaves and Pereira, 1992). However, studies of rice in response to both elevated [CO 2 ] and drought stress during the reproductive phase have not been conducted. Therefore, this study contains three objectives to investigate the effects of enhanced [CO 2 ] and water regimes on

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27 photosynthesis and photosynthate partitioning of rice leaves at panicle initiation and/or anthesis. Objective 1. To examine how leaf photosynthesis, total soluble protein and total chlorophyll in leaves is affected by water stress and elevated CO 2 Objective 2. To study effects of elevated-CO 2 and water stress on total non-structural carbohydrates in leaves. Objective 3. To determine sucrose phosphate synthase activity in leaves affected by water stress and CO 2 -enrichment.

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CHAPTER 2 EFFECTS OF ELEVATED [CO 7 ] AND WATER DEFICIT ON RICE LEAF PHOTOSYNTHESIS, CHLOROPHYLL CONTENT AND LEAF SOLUBLE PROTEIN Introduction Atmospheric enrichment generally enhances photosynthesis and plant growth rates (Kimball, 1983) Baker and Allen (1993) found that the direct effect of [CO 2 ] enrichment on rice (Oryza sativa L.), soybean (Glycine max L.) and citrus (various species) was always an increase i n photosynthetic rate. Furthermore, it was reported that photosynthetic rate of white clover (Trifolium repens L.) (Ryle et al. 1992a) and perennial ryegrass (Lolium perenne L. cv. Melle) (Ryle et al. 1992b) increased by 17-29% and 35-46%, respectively, with elevated (680 ppmv) [CO 2 ] treatment compared to 340 ppmv [CO 2 ] Arp and Drake (1991), studying Scirpus olneyi grown in elevated CO 2 found an increase in photosynthetic capacity by 31%. Short-term and long-term CO 2 enrichment of soybean plants resulted in increased rates of leaf photosynthesis (Huber et al., 1984; Valle et al., 1985; Campbell et al., 1990) and canopy photosynthesis (Jones et al., 1984a). Three seed crops, two forage crops and two native plant ecosystems grown in elevated CO 2 demonstrated increased canopy photosynthesis (Drake and Leadley, 1991). 28

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29 Baker et al. (1990b) and Rowland-Bamford et al. (1991) reported that rice photosynthetic rates increased with increasing [CO 2 ] treatment from 160 to 500 ppmv followed by a leveling off of the response among the superambient [CO 2 ] treatments. Rice photosynthetic and growth response to elevated CO 2 is reported to be highly dependent on the supply of N. Moreover, if additional CO 2 is given and N is not available, lack of sinks for excess carbon (e.g. tillers) may limit the photosynthetic and growth response (Ziska et al., 1996). Total soluble protein and leaf chlorophyll content of three-week-old soybean were reported to be unaffected by 70 Pa and 35 Pa CO 2 treatments (Sicher et al., 1995). Radoglou and Jarvis (1992) as well as Graham and Nobel (1996) reported that chlorophyll content per unit leaf area was less under elevated [CO 2 ] for Phaseolus vulgaris and the CAM plant, Agave deserti, respectively. Rowland-Bamford et al. (1991) reported that rice leaf nitrogen content decreased and rubisco protein as a fraction of total soluble leaf protein decreased by 32% with elevated CO 2 Water stress decreases net photosynthetic rate in crop plants and this decrease has previously been attributed largely to a decrease in stomata! conductance which restricts exogenous CO 2 supply and thereby changes the balance between carboxylation and oxygenation by Rubisco (Boyer and McPherson, 1981; Farquhar and Sharkey, 1982). In contrast, Di Marco et

PAGE 41

30 al. (1988) studying wheat (Triticum durum L.) concluded that CO 2 supply was not limiting, because the ratio of intercellular [CO 2 ] (C 1 ) to ambient [CO 2 ] (C a ) for the stressed plants was similar to the irrigated control. Also, the maximal rate of photosynthesis in saturating CO 2 of stressed pla n ts was quite similar to the rate of photosynthesis under natural conditions. Allen et al. (1994) also showed that the C t /C a ratio of soybean leaves did not change throughout a drought cycle. Although CO 2 was not limiting, Irigoyen et al. (1992) reported leaf water potential lower than -2.8 MPa directly affected CO 2 fixation. Water-stressed plants have lower leaf water potential and stomatal conductance. Further stress causes leaves to desiccate and therefore photosynthesis becomes inhibited and can be affected enough so that net carbon dioxide fixation ceases completely (Boyer and McPherson, 1981). Pea (Pisum sativum L. cv. Frilene) plants subjected to drought (Total leaf water potential=-1.3 MPa) were reported to have major reductions in photosynthesis (78%) and minor reduction (~18%) in the contents of chlorophyll a, carotenoid, and soluble protein (Moran et al., 1994). Furthermore, two cul ti vars of bean { Phaseol us vulgar is L.) decreased in soluble protein and chlorophyll content after drought stress was applied. In that study, chlorophyll content increased above control value and protein content also increased during the re-watering period (Castrillo and Trujillo, 1994).

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31 In drying soil, maize (Zea mays L.) photosynthesis became responsive to high CO 2 thus resulting in a considerable increase in dry matter and leaf area (Samarakoon and Gifford, 1996b). Gifford (1979) studying drought stressed wheat, found that elevated [CO 2 ] increased dry matter production due to increased water-use efficiency and resulted in adaptation to water stress through osmoregulation. Chaudhuri et al. (1986) reported that winter wheat under either drought stress or full irrigation and high [CO 2 ] has higher chlorophyll content compared to those of low [CO 2 ]. Little is known about rice responses to the combination of CO 2 enrichment and water deficit during the reproductive period, but it is a C 3 species whose photosynthesis is responsive to increases in [CO 2 ] (Baker et al., 1996a, 1996b; Ziska et al., 1996), and rice, however, is very sensitive to water stress during the reproductive phase (Yoshida, 1978; Baker et al., 1996a, 1996b). The objectives of this study were to determine the responses of rice photosynthesis under various water management treatments in long-term doubling of [COJ. Materials and Methods Plant material. The research was conducted during the 1994 growing season (15 July to 24 November) at the Irrigation Research and Education Park of the University of Florida at Gainesville, Florida (Baker et al. 199 6a, 19 9 6b) Rice

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32 (Oryza sativa L. cv. IR-72) plants were grown in eight sunlit controlled environment chambers. The chambers consisted of clear "sixlight" tops and walls 2.0 by 1.0 min cross section by 1.5 min height, attached to 0 6 m deep heavy-gauge bins filled with soil to a depth of 0 5 m. The soil used was fine sand (loamy, siliceous, hyperthermic Grossarenic Paleudult). A detailed description of the growth chamber design and computer control system may be found in Jones et al. (1984a, 1984b) while Baker et al. (1990a, 1990b) provide information on the modifications for growing rice. Pickering et al. (1993) described the current chamber system. The chambers were exposed to natural sunlight. Four chambers were controlled at 350 L CO 2 L 1 and 4 chamber were controlled at 700 L CO 2 L 1 In each pair of chambers (one ambient and one elevated [CO 2 ]), the following water management-stress regimes were imposed: a) continuously flooded (FLD), b) paddy flood water removed (soil bins drained) and drought stress imposed during panicle initiation (DPI), c) drought stress imposed during anthesis (DAN), and d) drought stress imposed at both panicle initiation and anthesis (DBS) (Table 2.1). Dry bulb air temperature and dew point were controlled to 28/21 (day/night) and to 18/12 c (day/night), respectively. Seeds of rice (cv. IR-72) were sown at rate of 200/m 2 in 11 rows 18 cm apart on 15 July 1994. After the seeds germinated and rice plants had one leaf, flood water was added daily in increments of 1 cm above the soil surface until water

PAGE 44

33 achieved a height of 5 cm where it was maintained. When rice plants reached growth stages for initiating drought treatments listed above, then paddy water was drained. When rice leaves started curling and desicating to the point of zero canopy assimilation, water was restored as shown in Table 2 .1. Further information on whole-canopy photosynthesis and evaporation during the imposed droughts, and growth and yield are given by Baker et al. (1996a, 1996b). Because the soil bins provided an absolute boundary and rice roots permeated the entire volume, expression of severe drought came suddenly and swiftly to the plants as the soil bins ran out of plant available soil water. It is important to note that the drought stress cycles were initiated by draining paddy water from the bottom of the soil bins. At the early stages of the imposed drought cycle, the soil was essentially in equilibrium with a very shallow water table (about 1-cm deep) at the bottom of the soil bins. Tensiometers in the soil did not indicate large soil water tensions until near the end of the drought cycle (Baker et al., 1996a, 1996b). Photosynthetic measurements. Photosynthetic measurement was conducted on three fully-expanded leaves from each chamber begining at 12:00 noon eastern standard time. Measurements were taken on various dates before drought, during drought and after re-watering. The LICOR LI-6250 Portable Photosynthesis Sys tern with O. 2 5 L chamber were used to measure photosynthetic rate at the corresponding CO 2 concentrations.

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34 Each data was computed from 3 measurements which each measurement consisted of 2 data. Table 2.1. Times when water was withheld and restored for drought treatments for all chambers. Rice was planted on 15 July 1994. CO 2 WM Stress at p. I. Stress WW WR WW L L 1 DAP 350 FLD t t t DPI 57 74 t DAN t t 93 DBS 57 74 96 700 FLD t t t DPI 57 75 t DAN t t 93 DBS 57 75 96 Note: FLD: continuously flooded DPI: drought imposed during panicle initiation DAN: drought imposed during anthesis DBS: drought imposed during both stages DAP: days after planting WM water management WW water withdrawn WR water restored t paddy water still present PI Panicle Initiation Total soluble protein. For soluble at Anthesis WR DAP t t 110 111 t t 110 111 protein and chlorophyll concentration analysis, 15 fully-expanded leaves were excised and rapidly immersed in liquid N 2 around 12:00 noon eastern standard time on various dates before drought, during drought and after re-watering. These same leaf samples were used for sucrose phosphate synthase analysis in Chapter 4. Leaves were collected from both the eastern half and western half of each chamber and combined.

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35 Leaf samples were preserved in liquid nitrogen until analysis. For soluble protein and chlorophyll assay, 0.1 g of frozen leaf was weighed and ground in chilled mortar using a 1:5 tissue-to-buffer ratio in a medium containing 5 ml of MOPS buffer at pH 7.5. After centrifugation, total soluble protein in an aliquot of the extract was determined with the dye binding method (Bradford, 1976) using gamma globulin as the standard. For soluble protein and chlorophyll, each data was calculated from two grinds out of one pooled sample. Each grind was divided for two extractions and each extraction was used for four assays. Leaf chlorophyll content. The leaf extract obtained from protein analysis above was used to measure chlorophyll concentration. Chlorophyll was extracted by diluting the extract in 80% acetone for 15 min in the dark at 4 c. After centrifugation, the chlorophyll content was determined using the method of Yoshida et al. (1972), measuring the absorbance at 645, 652 and 663 nm using a spectrophotometer. Results and Discussion Photosynthetic rate (Pn). The photosynthetic rate (Pn) of leaves of rice grown at 350 and 700 l CO 2 1 1 and in various water management treatments are given in Tables 2.2 through 2.5. The CO 2 -enriched plants exhibited higher Pn at each sampling date throughout the season. Before drought was imposed, the treatment differences were only attributed to CO 2

PAGE 47

level. Under continuously-flooding, the treatments increased leaf photosynthetic rate 36 doub 1 ed CO 2 by 40% from panicle initiation right through to final measurement before harvest (127 DAP). The results obtained in this study indicate that CO 2 enrichment increased photosynthetic rate (Pn) throughout the season in rice leaves when measured under continuously flooded water management. Increase of 50% in photosynthetic rate as a result of elevated CO 2 has been reported for rice plants, exposed to 660 compared to 330 bar (Ziska and Teramura, 1992b; Baker et al., 1990b). Balaguer et al. (1995) and Jacob et al. (1995) also observed increases of 47 and 45% in Pn as a result of doubled-CO 2 for Tri ticum aestivum and Scirpus olneyi, respectively. The increases in Pn between the elevated and ambient CO 2 treatment were slightly less pronounced at the end of the growing season. On the date of peak water deficit during the panicle initiation phase, leaf photosynthetic rate was decreased by 53% in enriched-CO 2 and 96% in ambient CO 2 plants (at 74 DAP), while water deficit during anthesis decreased Pn by 89% in enriched-CO 2 and 97% in low-CO 2 plants (at 114 DAP). A decrease was calculated as the difference between continuously-flooded treatment and corresponding water deficit and [CO 2 ] treatments (Table 2.3 and 2.5). At 72 DAP (Table 2.2), 11 days after water was removed, drought had already affected leaf photosynthetic rate (Pn) of

PAGE 48

37 plants grown at low CO 2 but drought had not affected Pn of the high CO 2 treatment. At 74 DAP, plants grown at 700 L co. L 1 still maintained moderate Pn (14. 7 and 10. 6 umol CO 2 m 2 s 1 ) while leaves grown at ambient CO 2 had very low Pn (0.9 and 3.8 umol CO 2 m 2 s 1 ). After re-watering at the end of day 74 (Table 2.3), plants grown at low [CO 2 ] required a longer time to recuperate from drought stress. At 84 DAP (Table 2.3), the 700DPI treatment had reached the Pn rate of 700FLD, but the Pn rate of the 350DPI treatment was still below the Pn rate of 350FLD. The lingering effect of drought on Pn rate seemed to continue through 89 OAP and perhaps 98 DAP (Table 2.4). At 109 DAP (Table 2.5), 16 days after water was withheld in 350DAN and 700DAN, photosynthetic rate of stressed leaves in ambient CO 2 was significantly lower than the rate of leaves of stressed plants in elevated CO 2 At the same time, 350D8S and 700D8S still maintained higher Pn compared to the two previous treatments. This may be due to 3-day delay in withholding water in 350DBS and 700DBS treatments. By 114 OAP (Table 2.5), 4 days after water was restored in 350DAN and 700DAN treatments and 3 days after water was restored in 350D8S and 700D8S treatments, Pn of the later treatments was significantly higher than the rate of former treatments. Ability of plants grown in high CO 2 to maintain high Pn after imposing water deficit was due to the effects of high CO 2 on water use efficiency (WUE). A number of studies have shown that elevated CO 2 reduces transpiration, primarily

PAGE 49

38 through a decrease in stomatal conductance (Valle et al., 1985; Rogers et al., 1992). Baker et al. (1990b) reported a decrease in evapotranspiration and found increases in WUE with increasing [CO 2 ] in rice. Rogers et al. (1992) reported that water use efficiency (the ratio of C gain to water loss) increased substantially in elevated CO 2 concentration. Leaf soluble protein concentration. Leaf soluble protein from rice grown at 350 and 700 L CO 2 L 1 and in various water management treatments is listed in Table 2.6 and 2.7. Leaf soluble protein of CO 2 -enhanced plants were significantly lower compared to those grown at ambient CO 2 treatment at 54, 82, 100, 110, and 127 DAP (P<0.05). The largest difference of leaf soluble protein concentration of plants grown in high-CO 2 and ambient-CO 2 chambers under continuously flooded treatment was by 18%. Rowland-Bamford et al. (1991) observed a 32% decrease in leaf soluble protein of rice with CO 2 enrichment and Jacob et al. (1995) reported a 45% decrease in soluble protein of Ficus opuntia. However, no effects of CO 2 -enrichment on leaf soluble protein were reported on 3-week-old soybean plants grown in 35 Pa and 70 Pa CO 2 (Sieber et al., 1995) and 58 day old soybean plants grown in 330, 450 and 600 mol mo1 1 (Allen et al., 1988). Table 2.6 and 2.7 show that water stress effects on leaf soluble protein concentration were significant at 74, 82, 92, 110 and 127 DAP. After paddy water was removed from 350DPI,

PAGE 50

39 3500BS, 700DPI and 700D8S treatments at 57 OAP to initiate stress treatment at panicle initiation, a decrease in leaf soluble protein concentration was detected at 74 OAP (Table 2.6) in elevated-CO 2 plants and ambient-CO 2 plants. The decrease was 62 and 56% in 3500PI and 7000PI treatments, respectively. Water was restored at 74 DAP in 3500PI and 35008S treatments and at 75 OAP in 7000PI and 700DBS treatments. By 92 OAP the protein concentration of the stressed plants had reached the level of leaf soluble protein concentration of continuously-flooded treatments (Table 2.6). For stress treatment at anthesis, paddy water was drained from chambers at 93 OAP for treatments of 350DAN and 700DAN, and at 96 OAP for treatments 35008S and 700D8S. By 100 OAP (Table 2.7), leaf soluble protein concentration was not yet affected by water stress treatment (P=0.5474). By 110 DAP the water management treatments were significantly different (P<0.01) and a decrease in leaf soluble protein concentration was detected (Table 2. 7) The decrease in leaf soluble protein content was 62 and 55% in 350DAN and 7000AN, and 56 and 53% in 350D8S and 700DBS, respectively. Leaf Chlorophyll Concentration. Leaf chlorophyll concentration of rice leaves grown at 350 and 700 uL CO 2 L 1 and for various water management treatments are given in Table 2. 8 and 2. 9. Chlorophyll concentration was significantly different with [CO 2 ] treatment from 54 to 110 OAP (P<0.05).

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In addition, the water management treatments significantly different at 74 and 110 OAP (P<0.05). 40 were Average increase chlorophyll concentration for enrichedCO 2 over ambient-CO 2 plants was detected under continuously flooded water treatment by 13%, but there was no increase at 110 nor 127 OAP (Table 2 9) Decreases in chlorophyll concentration of 62 and 73% in ambient-CO 2 and 48 and 54% in elevated-CO 2 leaves were observed during the water stress period at panicle initiation (74 OAP) and anthesis (110 DAP), respectively. Repeated water stress effects were significant and decreased chlorophyll concentration by 70% in low-CO 2 and 55% in doubled-CO 2 plants. Increase in leaf chlorophyll content of plants grown in elevated-CO 2 has also been found in soybean leaf blades (Allen at al., 1988). In addition, increase in chloroplast density or volume has also been reported to occur in soybean plants grown in an elevated CO 2 environment (Thomas and Harvey, 1983; vu et al., 1989). BY contrast, decrease of chlorophyll content of elevated CO 2 plants has been observed on pea (Xu et al., 1994), soybean (Radaglou and Jarvis, 1992), tomatoes (Khavari-Nejad, 1986) and other species (Delucia et al., 1985). Water deficit caused major reduction of chlorophyll concentration at panicle initiation at 74 OAP (Table 2.8). Decreases in chlorophyll were 62% and 48% in 350DPI and 700DPI plants, respectively. Reductions due to water stress at

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41 anthesis, 110 DAP (Table 2.8), were 73 and 76% in 350DAN and 350DBS, and 54 and 63% in 700DAN and 700DBS treatments, respectively. The decrease in chlorophyll content after drought stress has been reported on wheat (Chaudhuri et al., 1986), however, after re-watering, the chlorophyll content increased above control value (Castrillo and Trujillo, 1994). Based on the above results, doubled-[CO 2 ] treatments in rice had significantly increased leaf Pn and chlorophyll concentration, but decreased leaf soluble protein concentration. Withholding water during either panicle initiation or anthesis decreased Pn, leaf soluble protein and chlorophyll concentration.

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42 Table 2.2. Leaf photosynthetic rate of rice plants grown at 350 and 700 JIL CO 2 L 1 and in four different water managements. Measurements were taken on 51, 55, and 60 day-old plants (before stress affected} and 72 day-old plants (early water deficit period). Photosynthesis was measured with the LICOR LI-6250 using 0.25-L chamber with corresponding CO 2 concentrations. Water Leaf Photosynthetic Rate lJL L 1 350 700 Note: Management FLO DPI DAN DBS FLO DPI DAN DBS 51DAP ------23.8.0 24-0.4 23.9.1 22.2.9 30.4.9 30.5.5 32 2.5 30.3.9 P=0.0001 P=0.1417 P=0.6430 FLO: continuously flooded 55DAP 60DAP lJITIOl CO 2 m 2 s t 24.3.2 27.3.5 25.3.4 26.8.2 33.4.0 36.0.1 32.2.0 32.1.4 P=0.0001 P=0.0740 P=0.3962 24.3.7 27.1.1 26.0.6 23.4.2 34.8.2 32.7.9 28.6.2 26.4.7 P=0.0024 P=0 1121 P=0. 2713 DPI: drought imposed during panicle initiation DAN: drought imposed during anthesis DBS: drought imposed during both stages DAP: days after planting WM: water management 72DAP -------25.4.1 24.7.6 24.7.5 18.8.3 36.5.5 29.5.3 41.2.3 24.6.8 P=0.0001 P=0.0001 P=0.0264 Paddy water was removed at 57 OAP to lead to water stress at panicle initiation in 350DPI, 350DBS, 700DPI and 700DBS treatments. Water was restored after sampling at 74 DAP in 350DPI and 350DBS treatments, and at 75 OAP in 700DPI and 700DBS treatments.

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43 Table 2.3. Leaf photosynthetic rate of rice plants grown at 350 and 700 L CO 2 L 1 and in four different water managements. Measurements were taken at noon eastern standard time from 74 (stressed), 75 (stressed), 82 and 84 (re c overy) day-old plants. Photosynthesis was measured with the LICOR LI-6250 using 0.25L chamber with corresponding CO 2 concentrations. On 75 DAP, treatment 350DPI and 350DBS had been re-watered but 700DPI and 700DBS had not yet been watered. L L 1 350 700 Note: Water Management FLD DPI DAN DBS FLD DPI DAN DBS 74DAP 22.2.4 0.9.2 24.1.9 3.8.0 31.1.5 14.7.7 30.3.0 10.6.0 P=0.0001 P=0.0001 P=0.0692 FLD: continuously flooded Leaf Photosynthetic 75DAP 82DAP mol CO 2 m 2 s 1 20.1.8 12.5.9 21.5.2 13.5.3 28.8.8 1.1. 5 27.2.7 5.4.9 P=0.0015 P=0.0001 P=0.0001 23.7.0 17.3.1 22. 4. 4 17.9.3 33.3.7 30.6.3 31.5.2 29.6.4 P=0.0001 P=0.0005 P=0.0233 DPI: drought imposed during panicle initiation DAN: drought imposed during anthesis DBS: drought imposed during both stages DAP: days after planting WM: water management Rate 84DAP 21.9.0 17.2.3 23.1.9 18.9.5 33.9.6 33.0.7 34. 0. 2 32.9.7 P=0.0001 P=0.0818 P=0.0958 Water was restored after sampling at 74 DAP in 350DPI and 350DBS treatments, and at 75 DAP in 700DPI and 700DBS treatments.

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44 Table 2.4. Leaf photosynthetic rate of rice plants grown at 350 and 700 L CO 2 L 1 and in four different water managements. Measurements were taken on 89, 98 and 100 day-old plants. Photosynthesis was measured with the LICOR-6250 using 0.25-L chamber with corresponding CO 2 concentrations. L L 1 350 700 Water Management FLD DPI DAN DBS FLD DPI DAN DBS Leaf 89DAP -------mol 24.2.7 18.0.8 26.5.0 18.3.2 35.4.8 36.9.6 34.8.4 35.8.2 Photosynthetic Rate 98DAP l00DAP CO 2 m 2 s 1 22.6.8 25.8.8 20.4.5 27.1.4 24.6.1 24.2.4 16.1.7 23.2.5 33.0.8 33.4.3 36.5.8 35.1.9 37.6.7 31.1.3 33.2.5 30.5.4 Note: P=0.0001 P=0.0003 P=0.0001 FLD: continuously flooded P=0.0001 P=0.0001 P=0.0134 DPI: drought imposed during panicle initiation DAN: drought imposed during anthesis DBS: drought imposed during both stages DAP: days after planting WM: water management P=0.0001 P=0.0031 P=0.9599 Paddy water was removed at 93 DAP in 350DAN and 700DAN treatments, and at 96 DAP in 350DBS and 700DBS treatments. Water was restored at 110 DAP after sampling in 350DAN and 700DAN treatments, and at 111 DAP in 350DBS and 700DBS treatments.

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45 Table 2.5. Leaf photosynthetic rate of rice plants grown at 350 and 700 UL CO 2 L 1 and in four different water managements. Measurements were taken from 109, 114 and 125 day old plants. Photosynthesis was measured with LICOR-6250 using 0.25-L chamber with corresponding CO 2 concentrations. L L 1 350 700 Note: Water Management FLD DPI DAN DBS FLD DPI DAN DBS 109DAP 27.2.8 30.0.6 3.0.5 10.5.2 40.8.6 41.4.9 13.1.7 19.6.3 P=0.0001 P=0.0001 P=0.0223 FLD: continuously flooded Leaf mol Photosynthetic 114DAP CO 2 m 2 29.6.4 28.7.9 1.0.9 20.7.2 37.5.1 39.5.6 4.0.9 34.3.5 P=0.0001 P=0.0001 P=0.0002 s 1 DPI: drought imposed during panicle initiation DAN: drought imposed during anthesis DBS: drought imposed during both stages DAP: days after planting WM: water management Rate 125DAP 24.3.3 21. 2. 2 19. 2. 2 23.2.7 31.6.5 35.7.4 33.6.1 27.3.3 P=0.0001 P=0.2603 P=0.0188 Paddy water was removed at 93 DAP in 350DAN and 700DAN treatments, and at 96 DAP in 350DBS and 700DBS treatments. Water was restored at 110 DAP after sampling in 350DAN and 700DAN treatments, and at 111 DAP in 350DBS and 700DBS treatments.

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46 Table 2.6. Leaf soluble protein concentration of rice plants grown at 350 and 700 L CO 2 L 1 and in four different water managements Measurements were taken from 54, 74, 82 and 92 day-old plants. Water Leaf Soluble Protein Concentration L L 1 350 700 Note: Management FLD DPI DAN DBS FLD DPI DAN DBS 54DAP 37.2.7 43.0.8 37.5.3 35.7.1 30 4.3 30.8.4 33.1.5 30.0.5 P=0.0001 P=0.1119 P=0.1188 FLD: continuously flooded 74DAP 82DAP mg g l fresh weight 31.9.1 12.2.2 34.5 6 12.7.1 30.6.5 13.4 7 30.3.6 15.6.2 P=0.6115 P=0.0001 P=0.0100 30.4.5 28.9.9 32.4.6 29.3.9 30.8.6 24 4 8 32.2.0 26.1.7 P=0.0320 P=0.0005 P=0.1369 DPI: drought imposed during panicle initiation DAN: drought imposed during anthesis DBS: drought imposed during both stages DAP: days after planting WM: water management 92DAP 31.2.7 30.1.2 32.9.9 30.8.2 30.3.0 28 2.7 32.5.3 29.7.9 P=0.0865 P=0.0116 P=0.9277 Paddy water was removed at 57 DAP to lead to water stress at panicle initiation in 350DPI, 350DBS, 700DPI and 700DBS treatments. Water was restored after sampling at 74 DAP in 350DPI and 350DBS treatments, and at 75 DAP in 700DPI and 700DBS treatments

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47 Table 2.7. Leaf soluble protein concentration of rice plants grown at 350 and 700 uL CO 2 L I and in four different water managements. Measurements were taken from 100, 110 and 127 day-old plants. Water Management Leaf Soluble Protein Concentration lOODAP llODAP 127DAP UL L 1 350 700 Note: FLD DPI DAN DBS FLO DPI DAN DBS CO 2 WM C0 2 *WM 31.5.3 34.9.1 31.9.0 33. 9. 9 29.7.5 28.1.1 28.3.2 28.2.8 P=0.0001 P=0.5474 P=0.1714 FLD: continuously flooded mg g 1 fresh weight 32 .8. 2 32 .5.0 12.5.6 14.5.7 30.9.0 29.3.7 13. 9 3 14.6.6 P=0 0021 P=0.0001 P=0.1081 DPI: drought imposed during panicle initiation DAN: drought imposed during anthesis DBS: drought imposed during both stages DAP: days after planting WM: water management 25.6.4 24.5.9 20.9.1 20.5 5 23.4.4 25.0.4 20 9.1 18.5.7 P=0.0445 P=0.0001 P=0.0703 Paddy water was removed at 93 OAP in 350DAN and 700DAN treatments, and at 96 OAP in 350D8S and 700D8S treatments. Water was restored after sampling at 110 DAP in 350DAN and 700DAN treatments, and at 111 OAP in 350DBS and 700DBS treatments.

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48 Table 2. 8. Leaf chlorophyll concentration of rice plants grown at 350 and 700 L CO 2 L 1 and in four different water managements. Leaf samples were taken from 54, 74, 82 and 92 day-old plants. Water leaf Chlorophyll Concentration L L 1 350 700 Note: Management FLD DPI DAN DBS FLD DPI DAN DBS 54DAP 4.1.2 3.8.2 4.1.1 3.8.1 4.7.2 4.1.1 4.1.6 3.9.1 P=0.0005 P=0.0001 P=0.0049 FLD: continuously flooded 74DAP 82DAP mg g 1 fresh weight 3.7.5 1.4.3 3.8.6 1.3.5 4.4.4 2.3.6 4.0.8 2.3.3 P=0.0004 P=0.0001 P=0.2514 3.3.1 3.3.6 3.7.2 3.3.9 4. 0. 2 4.0.8 4.1.4 4.0.7 P=0.0001 P=0.2837 P=0.4540 DPI: drought imposed during panicle initiation DAN: drought imposed during anthesis DBS: drought imposed during both stages DAP: days after planting WM: water management 92DAP 3.7.7 3.9.4 3.7.9 3.6.2 4. 0. 0 4.0.2 4.0.1 3.9.2 P=0.0185 P=0.3691 P=0.7771 Paddy water was removed at 57 DAP to lead to water stress at panicle initiation in 350DPI, 350DBS, 700DPI and 700DBS treatments. Water was restored after sampling at 74 DAP in 350DPI and 350DBS treatments, and at 75 DAP in 700DPI and 700DBS treatments.

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49 Table 2. 9. Leaf chlorophyll concentration of rice plants grown at 350 and 700 uL CO 2 L I and in four different water managements. Measurements were taken from 100, 110 and 127 day-old plants. CO 2 water Leaf Chlorophyll Concentration Management l00DAP ll0DAP 127DAP UL L 1 350 700 Note: FLO DPI DAN DBS FLD DPI DAN DBS 3.7.3 3.8.9 3.6.5 3.7.6 4.1.3 4.0.2 4.0.1 4.0.8 P=0.0192 P=0.8363 P=0.9521 FLO: continuously flooded mg g I fresh weight 4.1.5 3.8.2 1. 1. 6 1.0. 8 4.1.6 4.0.1 1.9. 3 1.5.6 P=0.0178 P=0.0001 P=0.2943 OPI: drought imposed during panicle initiation DAN: drought imposed during anthesis DBS: drought imposed during both stages OAP: days after planting WM: water management 2.8.3 2.9.3 2.7.2 2.8.5 2.8.2 2.5.2 2.8.2 2.9.4 P=0.7084 P=0.9105 P=0.3086 Paddy water was removed at 93 OAP in 350DAN and 7000AN treatments, and at 96 OAP in 350D8S and 700DBS treatments. Water was restored after sampling at 110 DAP in 350DAN and 700DAN treatments, and at 111 OAP in 350DBS and 7000B5 treatments.

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CHAPTER 3 RICE LEAF NON-STRUCTURAL CARBOHYDRATES IN RESPONSE TO ENHANCED CO 2 AND WATER STRESS Introduction Sucrose and starch are the principal end products of photosynthesis in most plants, and sucrose is the principal carbohydrate translocated from source to sink tissues. During the day, leaves accumulate sucrose and starch as well as export sucrose to the rest of the plant (Preiss, 1982; Stitt et al., 1987). Leaf starch is an insoluble polyglucan that is deposited during the day light hours exclusively in the chloroplast (Preiss, 1982). Conversely, sucrose is a water soluble disaccharide that is synthesized in the cytosol and is the main form of reduced carbon translocated from source leaves to developing growth centers of the plant (Avigad, 19 82) It is well established that sucrose synthesis is decreased when sucrose accumulates in spinach leaves (Stitt et al 1987); however, Foyer (1987) suggested that the reduction in the capacity for sucrose synthesis caused by the buildup of sucrose is still questionable. Accumulation of starch in the leaves is a commonly reported response for plants grown in elevated CO 2 (Arp, 1991). Plants such as soybean (Glycine max) (Havelka et al., 50

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51 1984; Allen et al., 1988), tomato (Lycopersicon esculentum) (Yelle et al. 1989), bean (Phaseolus vulgaris) (Hoddinot and Jolliffe, 1988) and rice (Rowland-Bamford et al., 1990) showed an increase in leaf starch content after long-term (days to months) treatment with elevated CO 2 Rice leaf starch concentration tended to level off above 500 mol mo1 1 CO 2 Short-term enhancement of [CO 2 ) was reported to increase the accumulation of starch within source leaves of soybean (Huber et al., 1984; Sharkey et al., 1985). In leaves of tomato under elevated CO 2 basal (morning) leaf starch content was higher and the daily change greater due to a prolonged duration of starch accumulation during the day (Yelle et al., 1989). Leaves of bean (Hoddinot and Jolliffe, 1988) and soybean (Allen et al., 1988) also have an increased basal starch content. These findings suggested that increasing concentration of CO 2 altered the diel pattern of deposition and mobilization of chloroplast starch. Such increases in leaf starch may be the cause of the increase in specific leaf weight often observed under conditions of elevated CO 2 (Ehret and Jollife, 1985). Another example is soybean which was reported to have greater specific leaf weight (SLW) in elevated-CO 2 -grown plants (Allen et al 1988; Campbell et al., 1988}, while basil (Ocimum basilicum} was observed to exhibit a 1.5 to 2-fold greater increase in SLW as a result of an increase in atmospheric CO 2 concentration from 400 to 1500 .L CO 2 L 1 (Holbrook et al.,

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52 1993). At 1500 L CO 2 L 1 starch accounted for up to 38% of the total SLW of basil compared to <10% of SLW of spinach leaves. Furthermore, basil leaves grown in enriched-CO 2 showed chlorotic symptoms, whereas spinach leaves did not exhibit chlorosis. Arp (1991) proposed that starch accumulation appears to be primarily a symptom of the imbalance between supply and demand of carbohydrates and does not represent a significant feedback mechanism on photosynthetic capacity. Plants that do not form large amounts of starch tend to accumulate sucrose during the day; however those species such as soybean that accumulate starch typically do not accumulate soluble sugar (Allen et al., 1988; Huber, 1989). Starch and soluble sugar levels in leaves of wild radish plants (Raphanus sativus x raphanistrum) increased with increasing atmospheric [CO 2 ], whereas specific leaf area and nitrogen concentration of leaves significantly decreased (Chu et al., 1992). Fructose and glucose accumulated to a greater extent than sucrose at high CO 2 and may have been utilized for synthesis of cell -wall components, contributing to higher specific leaf weight. The ratio of starch to sucrose concentration of soybean leaves (Allen et al., 1988) and rice leaves (Rowland-Bamford et al., 1990) was positively correlated with the CO 2 concentration. Similarly, the starch: sucrose ratio of bean leaves also showed a marked increase with increasing intercellular CO 2 partial pressure

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53 (Sharkey et al., 1985). Moreover, formation of leaf starch and sucrose of bean was linearly related to assimilation. Due to the higher level of assimilate buildup and lower stomatal conductance to water loss, crops grown under high CO 2 may have capacity to delay stress effects on metabolism for a few days (Allen, 1994). Water-stressed leaves have decreased rates of starch synthesis and increased synthesis of sugars (Morgan, 1984). The author sugested that it is possible that changes in enzyme activities of the pathway leading to starch synthesis play a substantial role in altering partitioning during stress. When attached, translocating P. vulgaris leaves were water stressed and partitioning was assayed by 10-min pulse labelling with 14 CO 2 starch accumulation was decreased by more than 75% (Vassey and Sharkey, 1989). The purpose of this research was to investigate the effects of elevated atmospheric CO 2 and water stress during panicle initiation and anthesis stages on sucrose, starch and fructose concentration of rice plant leaves. Materials and Methods Plant material. Plant material was collected from rice plants as described in Chapter 2. For carbohydrate analysis, fully expanded leaves were sampled at 12:00 noon eastern standard time on various dates before drought, during drought and after re-watering. From each chamber, half the

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54 leaves were collected from the eastern side and half from the western side. The dry weight was determined after oven-drying at 70 c for 48 hours. Oven-dried leaves were ground in a ball mill to a fine powder. Sugar and starch analysis. Sucrose and starch determinations were carried out as described by Boote (1974) with slight modification Oven-dried powdered leaf material (0.05 g) was extracted four times with 3 ml of 80% ethanol (v/v) at 95 c for 1-h. It was confirmed that this was sufficient to quantitatively extract the ethanol-soluble sugars in the plant material. After centrifugation, supernatants were pooled and brought to a total volume of 15 ml with 80% ethanol. Sucrose, as fructose equivalents in sucrose, was analyzed with the resorcinol method described by Roe (1934) with some modification. A 0.25-mL aliquot was brought to 1 ml with 0.75 mL of distilled water and then boiled for 10 min with 1 mL of lN N a OH to destroy free fructose. After cooling, 1 mL resorcinol (0.1%, w/v in 95% ethanol) and 7 ml of HCl (9N) were added, mixed and the t~bes incubated at ao 0 c for 8 min. samples were cooled and read in spectrophotometer at 520 run. Total fructose equivalents (free fructose plus fructose equivalents in sucrose) were determined by the resorcinol test as described above except samples were boiled with 1 mL distilled water instead of lN NaOH. Free fructose was calculated from the difference between total fructose equivalents minus fructose equivalents of sucrose and

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55 reported after multiplying by 0.5263 (molar ratio of fructose to sucrose) because sucrose was used as the standard. The pellet obtained after 80% ethanol extraction was dried overnight at 60 c. Four ml of phosphate buffer was added to the dried pellet. In addition, a plant sample standard, starch standards, and a blank were similarly treated. After adding 1 ml of dialyzed a amylase, the test tubes were incubated at 85 c for 1 h. After adding 5 ml of acetate buffer and 1 ml of enzyme mix (containing: amyloglucosidase, invertase, acetate buffer and water) the test-tubes were incubated in a shaking water bath at 48 c for 24 h. Following incubation, the samples were filtered and the glucose content in the supernatant was determined by the Nelson-Somogyi test (Spiro, 1966). The results were converted to starch equivalents by comparing standard starch samples and glucose standards run in parallel with the plant material. Results and Discussion Leaf sucrose concentration. Data of leaf sucrose concentration (mg g 1 DW) of rice grown at 350 and 700 l CO 2 1 1 and under four different water management are given in Table 3.1 through 3.3. Effects of [CO 2 ] were significantly different throughout the season. Water stress treatments were significantly different in sucrose level on dates when water deficit occurred. As described earlier, panicle initiation was begun at 57 DAP; drought stress at therefore drought

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56 stress was still absent and only [CO 2 ] effects were present at 55 and 58 DAP. The average increase in leaf sucrose concentration of double[CO 2 ] plants from 55 DAP through the end of season was 9% under the continuously flooded treatment. Increase in leaf sucrose concentration of high-CO 2 plants has been also reported in soybean plants (Allen et al., 1988; Sicher et al., 1995), and increases of 40% were reported for rice (Rowland-Bamford et al 1990). By 72 DAP (Table 3 .1), 15 days after flood water was drained to initiate water stress, water management treatments were significantly different and showed [CO 2 ] effects as well as the interaction of both water management and [CO 2 ]. By 74 DAP, plants for 350DPI and 350DBS treatments started leaf curling/desication and sucrose concentration of ambient-CO 2 plants decreased by 82% (Table 3.1). Due to ability of high CO 2 to maintain high leaf water potential, 700DPI and 700DBS treatments had higher sucrose concentration compared to 350[CO 2 ] plants. At this time, both the 350DPI and 350DPA treatments had been re-watered for 1 day, so that sucrose concentration increased at 75 DAP, whereas 700DPI AND 700DBS plants had reached their leaf curling/desication point on this day (re-watered after sampling on 75 DAP) and water deficit decreased leaf sucrose concentration of elevated-CO 2 plants by 86% (Table 3.2). At 100 DAP (Table 3.3), there were no significant water deficit effects on leaf sucrose concentration,but effects were

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57 significant at 109 DAP (P<0.01). Leaf sucrose concentration of 350DAN and 700DAN treatments was lower than the concentration of 3 50DBS and 700DBS treatments (Table 3. 3) possibly because DBS treatments senesed lower leaves during the previous drought cycle at panicle initiation, and had less transpiring surface (Baker et al., 1996a, 1996b). Minor effects of water deficit during anthesis carried through to 125 DAP (P<0.01). Decreases in sucrose concentration of stressed leaves by 69% in low-CO 2 and 58% in doubled-CO 2 plants were observed for DAN water deficit treatments. However, sucrose concentration was decreased only by 37% in low-CO 2 and 32% in elevated-CO 2 plants for rice that was exposed to water deficit at two stages of growth (DBS). Leaf starch concentration. Leaf starch concentration (mg g 1 DW) of rice plants grown at 350 and 700 L CO 2 L 1 and four different water regimes are given in Table 3.4 through 3. 6. The [CO 2 ) effects on leaf starch were significant during early reproductive stages (58 through 74 DAP) (P<0.05). Elevated-CO 2 plants had higher starch concentration before and after panicle initiation which occurred at 74 DAP, but after that starch concentration of high-CO 2 and ambient-CO 2 plants were similar. Increase in leaf starch concentration of elevated-CO 2 plants has also been reported for soybean (Allen et al., 1988), bean (Radaglou and Jarvis, 1992) and rice (Rowland Bamford et al., 1990). The difference in leaf starch and

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58 sucrose concentration between leaves grown at elevated and current CO 2 diminished with plant maturity. Water stress effects on starch concentration were significant at 72, 74, 75, 82, and 109 DAP as shown in Table 3.4 through 3.6. The CO 2 by water management interaction was significantly different only at 75 DAP. After paddy was drained from chambers (350DPI, 350DBS, 700DPI and 700DBS) at 57 DAP for stress treatment at panicle initiation, a decrease in leaf starch concentration was detected at 74 DAP (Table 3.4). Low-CO 2 plants (350DPI) were more quickly affected by water deficit treatment so that leaf starch concentration of that treatment decreased by 93%. By 75 DAP, leaves of high CO 2 plants were highly stressed and leaf starch concentration decreased by 92%. After water was restored to the drought treatment at 74 and 75 DAP for ambient-CO 2 and elevated-CO 2 respectively, the stressed plants of both [CO 2 ] treatments recovered to initial starch value by 89 DAP (Table 3. 8) so that there was no significant difference in water-treatment on that day. It is interesting to note that water-deficit-induced differences in leaf starch persisted for more than 7 days after water was restored (until at least 82 DAP). For stress treatment at anthesis, paddy water was drained from chambers at 93 DAP in treatments 350DAN and 700DAN, and at 96 DAP in 350DBS and 700DBS. At 100 DAP (Table 3.5), the leaf starch concentration was not affected by water stress

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59 treatment (P=0.11). By 109 DAP (Table 3.9), the decrease in leaf starch concentration was significant, and the water management treatments were significantly different (P<0.01). Decrease in leaf starch concentration due to water deficit has also been observed by Morgan (1984) and Vassey and Sharkey (1989). The later authors found 75% decrease in leaf starch concentration in bean (Phaseolus vulgaris). The decrease in leaf starch and sucrose of plants with increasing plant development has been observed on rice (Rowland-Bamford et al., 1990) and wheat (Nie et al., 1995). Decline in leaf starch and sucrose concentration might be associated with the onset of grain filling. Leaf fructose concentration. Leaf fructose concentrations in leaves from rice grown at 350 and 700 l CO 2 1 1 and in various water management treatments are listed in Table 3.7 through 3.9. Leaf fructose concentration of CO 2 enhanced plants were significantly higher compared to those grown at ambient CO 2 treatment throughout the season (P<0.01). Leaf fructose concentration of plants grown in high-CO 2 chambers under continuously flooded treatment was 28 to 92% higher than ambient-CO 2 plants for the various dates from panicle initiation (54 DAP) through to final harvest (127 DAP). Elevated CO 2 treatments had relatively higher fructose concentration early in the season (DAP 55 and 58) than later (DAP 72 and beyond).

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60 Water stress effects were significant at 74, 75, 82 and 109 OAP (Table 3.7, 3.8, and 3.9). At 74 and 109 OAP, elevated-CO 2 plants were able to maintain leaf fructose concentration relatively higher than those of low-CO 2 plants. After paddy water was drained from chambers (350OPI, 350OBS, 700OPI and 700OBS) at 57 OAP for stress treatment at panicle initiation, a decrease in leaf fructose concentration was detected at 74 OAP, and at that time, elevated-CO 2 plants were able to maintain leaf fructose concentration relatively higher than ambient-CO 2 plants. After water was restored at 74 and 75 OAP for ambient-CO 2 and elevated-CO 2 treatments, respectively, stressed plants of both [CO 2 ] treatments recovered to initial fructose concentration values by 92 OAP (Table 3.8). There was no significant effect of water-management treatment at 92 OAP. For stress treatment at anthesis, paddy water was drained from chambers at 93 OAP for treatments of 350OAN and 700OAN, and at 96 DAP for 350D8S and 700DBS treatments. At 100 DAP (Table 3.9), the leaf fructose concentration was not affected by water stress treatment (P=0.82), however by 109 DAP (Table 3.9), the decrease in leaf fructose was significant for the water treatment (P<0.01). Leaves of plants grown in high-CO 2 were able to maintain higher leaf fructose concentration and were able to recover more quickly than those of plants grown at low CO 2

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61 Elevated CO 2 treatments were able to avoid or compensate for water stress for 1 to 2 days longer, i.e., elevated-CO 2 plants were able to maintain moderate sucrose, starch and fructose concentration 1 to 2 days longer compared to ambient CO 2 plants. Elevated-CO 2 plants had ability to recover more quickly than ambient-CO 2 plants.

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62 Table 3.1. Leaf sucrose concentration of rice plants grown at 350 and 700 L CO 2 L 1 and in four different water managements. Leaf samples were taken at noon eastern standard time from 55, 58, 72 and 74 day-old plants. Water Leaf Sucrose Concentration L L 1 350 700 Note: Management FLO DPI DAN DBS FLO DPI DAN DBS 55DAP 82.0.0 84.2.7 83.3.5 85.3.0 95.0.2 92.8.4 93.4.2 90.0.0 P=0.0001 P=0.9744 P=0.3071 FLD: continuously flooded 58DAP 72DAP mg g l dry weight 73.1.7 73.7.2 74.6.0 75.6.6 85.3.4 87.4.5 86.4.6 85.5.6 P=0.0001 P=0.9658 P=0.9413 66.7.6 49.1.0 66.2.6 53.5.3 80.9.9 58.7.1 80.8.3 53.8.9 P=0.0001 P=0.0001 P=0.0170 DPI: drought imposed during panicle initiation DAN: drought imposed during anthesis DBS: drought imposed during both stages OAP: days after planting WM: water management 74DAP 62.6.8 9.4.9 63.4.7 12.6.5 78.3.9 28.3.7 74.4.6 28.3.2 P=0.0001 P=0.0001 P=0.7301 Paddy water was removed at 57 OAP to initiate water stress at panicle initiation in 350DPI, 350DBS, 700DPI and 700DBS treatments. Water was restored after sampling at 74 DAP in 350DPI and 350DBS treatments, and at 75 OAP in 700DPI and 700DBS treatments.

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63 Table 3.2. Leaf sucrose concentration of rice plants grown at 350 and 700 L CO 2 L 1 and in four different water managements. Leaf samples were taken at noon eastern standard time from 75 (stressed), 82 and 89 (recovery) day-old plants. Treatments 350DPI and 350DBS had been re-watered for 1 day, but 700DPI and 700DBS had not yet been watered. Water Leaf Sucrose Concentration L L 1 350 700 Note: Management FLD DPI DAN DBS FLD DPI DAN DBS 75DAP 59.6.3 28.7.8 59.0.9 22.2.6 65.9.6 9.6.6 68.7.6 7.7.8 P=0.0530 P=0.0001 P=0.0001 FLD: continuously flooded DPI: drought imposed during DAN: drought imposed during DPA: drought imposed during DAP: days after planting WM: water management mg g l 82DAP dry weight 59.2.4 51.0.2 58.2.8 52.8.0 60. 6. 5 51. 9. 4 60.3.8 52.6.4 P=0.6008 P=0.0162 P=0.9824 panicle initiation anthesis both stages 89DAP 59. 5. 3 61.1.4 58.9.3 64.5.4 58.1.9 63.7.7 59.6.8 63.0.6 P=0.9325 P=0.1256 P=0.7765 Water was restored after sampling at 74 350DBS treatments, and at 75 DAP in treatments. DAP in 350DPI and 700DPI and 700DBS

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64 Table 3.3. Leaf sucrose concentration of rice plants grown at 350 and 700 L CO 2 L 1 and in four different water managements. Leaf samples were taken at noon eastern standard time from 100, 109 (deficit period) and 125 (recovery period) day-old plants. Water Leaf Sucrose Concentration Management l00DAP 109DAP 125DAP L L 1 350 700 Note: FLD DPI DAN DBS FLD DPI DAN DBS 47.9.1 51.6.9 50.8.7 51.0.6 50.2.4 50.4.6 48.5.6 51. 5. 2 P=0.8763 P=0.2230 P=0.2629 FLD: continuously flooded mg g 1 dry weight 50.3.2 48.6.2 15.5.0 31.7.9 44.6.3 48 .4.1 18.9.8 30.5.8 P=0.0550 P=0.0001 P=0.0001 DPI: drought imposed during panicle initiation DAN: drought imposed during anthesis DPA: drought imposed during both stages DAP: days after planting WM: water management 39.1.8 41.8.8 35.4.2 36.3.3 37.4.2 40.5.7 34.3.2 34. 5.1 P=0.0105 P=0.0001 P=0.0537 Paddy water was removed at 93 DAP in 350DAN and 700DAN treatments, and at 96 DAP in 350DBS and 700DBS treatments. Water was restored after sampling at 110 DAP in 350DAN and 700DAN treatments, and at 111 DAP in 350DBS and 700DBS treatments.

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65 Table 3.4. Leaf starch concentration of rice plants grown at 350 and 700 L CO 2 L 1 and in four different water managements. Leaf samples were taken at noon eastern standard time from 55 58, 72 and 74 day-old plants. Water Leaf Starch Concentration L L 1 350 700 Note: Management FLD DPI DAN DBS FLD DPI DAN DBS 55DAP 54.5.0 56.7.9 55.3.5 51.4.2 76.8.4 78.9.4 71.9.3 75.4.4 P = 0.0001 P=0.5909 P=0.7419 FLD: continuously flooded 58DAP 72DAP mg g i dry weight 42.3.8 43.3.9 41.5.0 44.4.2 59.3.4 61. 4. 5 61.1. 7 63.3.3 P=0.0001 P=0.7728 P=0.9787 53.8.6 37.4.0 55.3.1 33.4.4 59. 5. 8 54.4.8 57.7.3 37.7.7 P=0.0030 P=0.0001 P=0.1037 DPI: drought imposed during panicle initiation DAN: drought imposed during anthesis DBS: drought imposed during both stages DAP: days after planting WM: water management 74DAP 46.3.2 3.4.4 47.3.5 3.6.5 51.1. 2 10.3.4 47.0.3 11. 7. 5 P=0.0024 P=0.0001 P=0.1683 Paddy water was removed at 57 DAP to initiate water stress at panicle initiation in 350DPI, 350DBS, 700DPI and 700DBS treatments. Water was restored after sampling at 74 DAP in 350DPI and 350DBS treatments, and at 75 DAP in 700DPI and 700DBS treatments.

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66 Table 3.5. Leaf starch concentration of rice plants grown at 350 and 700 L CO 2 L 1 and in four different water managements. Leaf samples were taken at noon eastern standard time from 75 (stressed), 82 and 89 (recovery) day-old plants. Treatments 350DPI and 350DBS had been re-watered for 1 day, but 700DPI and 700DBS had not yet been watered. Water Leaf Starch Concentration L L 1 350 700 Note: Management FLD DPI DAN DBS FLD DPI DAN DBS 75DAP 40.1.5 10.3.0 40.7.3 8.4.1 50.7.0 4.1.3 50.3.6 7.5.8 P=0.0860 P=0.0001 P=0.0103 FLD: continuously flooded mg g l 82DAP dry weight 21. 0. 5 12. 8. 9 25.3.2 11.4.0 24.7.1 13.5.3 22. 6. 8 14.3.8 P=0.4175 P=0.0001 P=0.4020 DPI: drought imposed during panicle initiation DAN: drought imposed during anthesis DPA: drought imposed during both stages DAP: days after planting WM: water management 89DAP 21. 7. 9 21.4.9 20.1.3 22. 8. 0 21.8.7 21.5.8 21.7.9 20.2.6 P=0.7964 P=0.9048 P=0.3998 Water was restored after sampling at 74 DAP in 350DPI and 350DBS treatments, and at 75 DAP in 700DPI and 700DBS treatments.

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67 Table 3.6. Leaf starch concentration of rice plants grown at 350 and 700 uL CO 2 L 1 and in four different water managements. Leaf samples were taken at noon eastern standard time from 100, 109 (stressed) and 125 (recovery) day-old plants. UL L 1 350 700 Note: Water Management FLD DPI DAN DBS FLD DPI DAN DBS Leaf l00DAP 11. 5. 4 11. 8. 6 10.9.9 12.7.5 13 4.2 12 9. 5 10.4.8 12.8.5 P=0.2979 P=0.1135 P=0.5881 FLD: continuously flooded Starch Concentration mg g l 109DAP dry weight 11.8.9 11. 7. 2 7.0.3 9.4.7 12.1.9 10.3.4 7. 8. 6 10.6.0 P=0.6194 P=0.0001 P=0.2438 DPI: drought imposed during panicle initiation DAN: drought imposed during anthesis DPA: drought imposed during both stages DAP: days after planting WM: water management 125DAP 12.5.4 12.7.0 11.3.9 10.0.5 11. 3.1 12.2.3 11.2.0 12.1.6 P=0.9207 P=0.6042 P=0.5021 Paddy water was removed at 93 DAP in 350DAN and 700DAN treatments, and at 96 DAP in 350DBS and 700DBS treatments. Water was restored after sampling at 110 DAP in 350DAN and 700DAN treatments, and at 111 DAP in 350DBS and 700DBS treatments.

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68 Table 3.7. Leaf fructose concentration of rice plants grown at 350 and 700 uL CO 2 L 1 and in four different water managements. Leaf samples were taken at noon eastern standard time from 55, 58, 72 and 74 day-old plants. Water Leaf Fructose Concentration UL L 1 350 700 Note: Management FLD DPI DAN DBS FLD DPI DAN DBS 55DAP 5.0.2 4. 2. 3 4.5.4 4.6.5 8.2.3 8.3.3 8.4.5 8.2.2 P=0.0001 P=0.2252 P=0.2296 FLD: continuously flooded 58DAP 72DAP mg g l dry weight 4 9.7 4.6.9 5.0.5 4.7.7 9.4.3 9.5.5 9.0.5 9.2.6 P=0.0001 P=0.9928 P=0.5426 8.8.8 5. 0. 6 8.7.8 4.7.3 11. 3. 2 11. 8. 5 11. 6. 6 12.7.8 P=0.0001 P=0.0139 P=0.0007 DPI: drought imposed during panicle initiation DAN: drought imposed during anthesis DBS: drought imposed during both stages DAP: days after planting WM: water management 74DAP 9.2.8 0.9.9 9 .2. 9 1.1.7 11. 9. 2 8.9.6 12.5.2 7.9.7 P=0.0001 P=0.0001 P=0 0001 Paddy water was removed at 57 DAP to initiate water stress at panicle initiation in 350DPI, 350DBS, 700DPI and 700DBS treatments. water was restored after sampling at 74 DAP in 350DPI and 350DBS treatments, and at 75 DAP in 700DPI and 700DBS treatments.

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69 Table 3.8. Leaf fructose concentration of rice plants grown at 350 and 700 L CO 2 L 1 and in four different water managements. Leaf samples were taken at noon eastern standard time from 75 (stressed), 82 and 89 (recovery) day-old plants. Treatments 350DPI and 350DBS had been re-watered for 1 day, but 700DPI and 700DBS had not yet been watered. L L 1 350 700 Note: Water Management FLD DPI DAN DBS FLD DPI DAN DBS 75DAP ------8.6.5 3.2.8 8.9.9 2.8.6 11.9.6 1.9.8 13.1.7 1. 6. 8 P=0.0034 P=0.0001 P=0.0001 FLD: continuously flooded DPI: drought imposed during DAN: drought imposed during DPA: drought imposed during DAP: days after planting WM: water management Leaf Fructose Concentration mg g l 82DAP dry weight 9.2.4 5.6.5 8.5.7 6. 4. 5 12.2.7 9.7.0 12.3.4 9.6.6 P=0.0001 P=0.0001 P=0.4380 panicle initiation anthesis both stages 89DAP 8.0.4 8.9.4 8.9.8 7.5.4 11. 0. 9 12.0.7 11.9.9 12.4.2 P=0.0001 P=0.6729 P=0.0302 Water was restored after sampling at 74 350DBS treatments, and at 75 DAP in treatments. DAP in 350DPI and 700DPI and 700DBS

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70 Table 3.9. Leaf fructose concentration of rice plants grown at 350 and 700 L CO 2 L 1 and in four different water managements. Leaf samples were taken at noon eastern standard time from 100, 109 (stressed) and 125 (recovery) day-old plants. Water Leaf Fructose Concentration Management L L l 350 FLD DPI DAN DBS 700 FLD DPI DAN DBS Note: l00DAP ------8.1.2 9.0.9 8.3.7 8.9.6 10.2.4 10.5.6 10. 7. 6 10. 5. 2 P=0.0001 P=0.8181 P=0.9037 FLD: continuously flooded mg g l 109DAP dry weight 7. 7. 2 7.8.4 1. 5. 0 3.8.9 9.6.3 9.4.1 5.3.8 7.7.3 P=0.0001 P=0.0001 P=0.0039 DPI: drought imposed during panicle initiation DAN: drought imposed during anthesis DPA: drought imposed during both stages DAP: days after planting WM: water management 125DAP 7.0.8 6.9.8 6.9.2 6.7.3 7. 9. 2 7.6.7 7.7.2 7.6.1 P=0.0001 P=0.2932 P=0.9681 Paddy water was removed at 93 DAP in 350DAN and 700DAN treatments, and at 96 DAP in 350DBS and 700DBS treatments. Water was restored after sampling at 110 DAP in 350DAN and 700DAN treatments, and at 111 DAP in 350DBS and 700DBS treatments.

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CHAPTER 4 THE EFFECTS OF ELEVATED CO 2 CONCENTRATION AND WATER STRESS ON SUCROSE PHOSPHATE SYNTHASE ACTIVITY Introduction Sucrose phosphate synthase (SPS) is a key enzyme in the rate-control of sucrose synthesis and its activity in a variety of species has been negatively correlated with leaf starch accumulation (Huber, 1981, 1983; Huber et al., 1984). Negative correlation between SPS activity and starch accumulation for CO 2 -enriched plants has observed for leaves of Cucumis sativus (Peet et al., 1986). Short-term treatment of soybean plants with elevated CO 2 doubled the starch content and decreased the activity of SPS within the leaves (Huber et al., 1982, 1984). In contrast, the SPS activity of rice leaves measured 59 dafter planting in 660 L L 1 CO 2 has been found to increase twoand three-fold when compared with the control (Hussain et al., 1990). Moreover, soybean plants grown in high-N media (20 mol m 3 KNO 3 ) have a higher rate of assimilate export, higher SPS activity, higher sucrose and decreased starch concentration within the leaves when compared to plants grown at 10 mol m 3 KNO 3 (Huber et al., 1984). Short-term treatment of high-N supplied plants with elevated CO 2 increased leaf starch 71

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72 concentration at the expense of export of assimilate from the leaf. Although the sucrose concentration of the leaf in this case was unchanged, there was a marked reduction of SPS activity (Huber et al., 1984). SPS plays an important role as the limiting factor of partitioning of carbon sources. Therefore, it seems that enhancement of SPS activity may promote the ability of the source function. Makino et al. (1994) demonstrated that CO 2 assimilation rates of young rice plants grown at ambient CO 2 levels were limited by SPS activity when blades were exposed to high CO 2 during gas exchange measurements. When plants are exposed continuously to high CO 2 synthesis of SPS may be increased to optimize photosynthesis and growth under this particular environment, thereby resulting in greater SPS activity. CO 2 enrichment increased SPS activity of the rice cultivar IR-30 when measurements were made at the mid tillering phase (Baker et al., 1988). In the same study, it was demonstrated that CO 2 enrichment had no effect on SPS activity of soybean leaves. In contrast to rice, soybean leaves accumulate large amounts of starch (Huber et al., 1982, 1984; Allen et al., 1988). The CO 2 -saturated photosynthesis was positively correlated with the activities of SPS in the high-N leaves. SPS activity increased steadily with increasing leaf nitrogen concentration (Makino et al., 1994).

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73 Seneewera et al. (1995) concluded that the enhancement of SPS activity by CO 2 enrichment in fully expanded leaf blades plays a major role in determining the capacity of these source blades to supply the growing sink with sucrose. In short term water deficit, the partitioning of newly fixed photosynthate favouring sucrose at the expense of starch is among the early effects of tissue dehydration in some species (Quick et al., 1989, 1992). This increased partitioning of recent assimilates to sucrose has been linked to alterations in sucrose phosphate synthase (SPS) activity and to a general increase in the amounts of metabolites due to a decrease in cytoplasmic volume under drought (Quick et al., 1989). Although some disagreements have been observed as a consequence of measurements being made with plants either under ambient or saturating CO 2 (Vassey and Sharkey, 1989; Vassey et al., 1991), it is likely that the preferential partitioning of recent assimilates to sucrose in water stressed leaves is accompanied by an increased activation of SPS in spinach (Zrenner and Stitt, 1991). Little is known about the effect of enhanced CO 2 concentration and water stress during panicle initiation and anthesis on the activities of sucrose phosphate synthase in leaves of rice. Therefore this study was conducted to evaluate possible interaction effects of elevated CO 2 and water stress on SPS activity of rice around panicle initiation and anthesis stages.

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74 Materials and Methods Plant materials. Plant material was collected from rice plants grown under conditions described in Chapter 2. For measurements of SPS activity, 15 fully-expanded leaves were excised and rapidly immersed in liquid N 2 around 12:00 noon eastern standard time on various dates before drought, during drought and after re-watering There were 8 sunlit, controlled-environment chambers of which 4 chambers were set at 350 L CO 2 L 1 and 4 chamber were set at 700 L CO 2 L 1 In each pair of chambers (one ambient and one elevated [CO 2 ]), the following water management-stress regimes were imposed: a) continuously flooded (FLD), b) paddy flood water removed and drought stress imposed during panicle initiation (DPI), c) drought stress imposed during anthesis (DAN), and d) drought stress imposed during both panicle initiation and anthesis (DBS). Leaves were collected from both the eastern half and the western half of each chamber and composited prior to storage in liquid N. Enzyme extraction. Leaf samples previously frozen and stored in liquid nitrogen were weighed (0.2 g) and ground in chilled mortar with 2.5 ml extraction buffer (50 mM MOPS-NaOH [pH 7.5], 15 mM MgC1 2 1 mM EDTA, 2.5 mM DTT, and 0.1% (v/v) Triton X-100). The extract was centrifuged in a 1.2-ml microtube at 13,000 g for 1.5 min. The supernatant solution was desalted by centrifugal gel filtration into buffer

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75 containing 50 mM MOPS-NaOH [pH 7.5), 15 mM MgCl 2 1 mM EDTA, and 2.5 mM DTT. Enzyme Assay. Sucrose phosphate synthase was measured in the synthetic direction by quantitation of sucrose formation using the resorcinol method. Assays were conducted by incubating 45 L of tissue extract for 10 min at 25 c with 10 mM UDPG, 10 mM F6P, 40 mM G6P, 50 mM MOPS-NaOH (pH 7.5), 15 mM MgC1 2 and 2.5 mM DTT in a total volume of 70 L. The reaction was terminated by the addition of 70 L 1 N NaOH. Tubes were boiled for 10 min to destroy any remaining fructose (or unreacted F6P), then 250 L 0.1% (w/v) resorcinol in 95% ethanol plus 750 L 30% (v/v) HCl were added. Tubes were incubated at 80 C for 8 min and after cooling, the A ~ 0 was measured with a Spectrophotometer. Sucrose formation was quantitated by comparison to a sucrose standard curve after subtraction of A 5 20 at O min (background) The background standard was handled essentially the same as described earlier except that 70 L lN NaOH was added to reaction mixture before adding extract tissue, so that the reaction would not occur. Results and discussion Sucrose phosphate synthase. Sucrose phosphate synthase activities in leaves from rice grown at 350 and 700 L CO 2 L 1 and in various water management treatment are listed in Table 4 .1 and 4. 2. SPS activities of CO 2 enhanced plants were significantly higher compared to those grown at ambient CO 2

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76 treatment throughout the season (P<0.05). SPS activities of plants grown in high CO 2 chambers under continuously-flooded treatment were consistenly higher than ambient CO 2 treatments by 6 to 16% from panicle initiation (54 DAP) through right before final harvest (127 DAP). The results obtained in this study indicate that CO 2 enrichment increased SPS activities throughout the season in rice leaves when measured under continuouslyflooded water management. Enhancement of SPS activity by elevated CO 2 levels has also been reported on rice (Baker et al., 1988; Hussain et al., 1990; Makino et al., 1994; and Seneweera et al., 1995). Huber et al. (1984), however, demonstrated that CO 2 enrichment had no effect on SPS activity of soybean leaves. Water stress effects were significant at 74 and 110 DAP (Table 4.1 and 4.2). On those dates, elevated-CO 2 plants were able to maintain SPS activity relatively higher than those of low-CO 2 plants. After paddy water was withdrawn from chambers (350DPI, 350DBS, 700DPI and 700DBS) at 57 DAP for stress treatment at panicle initiation, a decrease in SPS activity was detected at 74 DAP (Table 4 .1) Water management treatments were significantly different in SPS activities at 74 and 110 DAP (P<0.001). After water was restored for DPI and DBS treatments at 74 and 75 DAP for ambient-CO 2 and elevated-CO 2 respectively, plants of both [CO 2 ] treatments recovered to initial SPS activities by 82 and 92 DAP (Table 4.1), so that there was no

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77 significant difference of water-treatment on those recovery days. For stress treatment at anthesis, paddy water was drained from chambers at 93 DAP for treatments of 350DAN and 700DAN, and at 96 DAP for 350DBS and 700DBS treatments. At 100 DAP (Table 4.2), the effect of water stress treatment was not yet significantly different (P=0.57). A decrease in SPS activity was detected at 110 DAP, when the water deficit treatment was significantly lower (P<0.01). Studies of water stress effects on sucrose phosphate synthase (SPS) activities have also been studied on soybean (Quick et al., 1989, 1992), Phaseolus vulgaris (Vassey and Sharkey, 1989; Vassey et al., 1991), and spinach (Zrenner and Stitt, 1991). However, they have failed to reach a consensus (Quick et al., 1989; Vassey and Sharkey, 1989). In soybean, water-stressed leaves had SPS activities that were not significantly different from controls, thus this element of export capacity was maintained. However, in Phaseolus vulgar is leaves, mild water stress ( total leaf water potential at about -1.0 MPa) inhibited SPS by more than 60% (Vassey and Sharkey, 1989). A study of spinach leaves showed that water stress had differing effects on two kinetic forms of SPS, detected by assay conditions (Quick et al., 1989). With "nonselective" assay conditions, where substrate concentration was high and orthophosphate (P 1 ) was omitted, there was no significant

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78 effect of water stress on SPS activity. With "selective" assay conditions, where P i was included to inactivate the kinetically-inactive form of the enzyme and reveal the kinetically-active form, a substantial increase in SPS activity was found as total leaf water potential was decreased from O to -1 2 MPa. The previously mentioned studies of soybean and P. vulgaris used assay conditions that were nonselective. Thus, the extent to which changes in SPS activity contribute to changes in partitioning between sucrose and starch may depend on the relative co ntribution of these kinetic forms to SPS activity as it exists in vivo.

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79 Table 4 .1. Sucrose phosphate synthase activities of rice plants grown at 350 and 700 L CO 2 L 1 and in four different water managements. Plant samples were taken at noon eastern standard time from 54, 74 (stressed), 82 and 92 (recovery) day-old plants. L L 1 350 700 Note: Water Management FLD DPI DAN DBS FLD DPI DAN DBS 54DAP mol 49.7.1 50.2.9 50.2.7 50.5.5 55.2.7 55.2.5 54.8.4 55.4.4 P=0.0014 P=0.9866 P=0.9882 FLD: continuously flooded SPS Activities 74DAP (g fresh 47.6.5 13.4.3 48.6.6 15.3.1 54.8.1 23.2.5 62.3.6 21.9.2 P=0.0047 P=0.0001 P=0.6898 82DAP weight) 1 47.1.4 46.6.7 47.1.4 46.6.7 51.3.1 50.0.8 50.8.6 51.0.8 P=0.0126 P=0.9615 P=0.9904 ... DPI: drought imposed during panicle initiation DAN: drought imposed during anthesis DBS: drought imposed during both stages DAP: days after planting WM: water management 92DAP h l 44.4.2 44.8.9 44.9.3 45.8.1 49.2.8 49.0.6 49.7.7 49.5.1 P=0.0082 P=0.9608 P=0.9852 Paddy water was removed at 5"7 DAP to initiate water stress at panicle initiation in 350DPI, 350DBS, 700DPI and 700DBS treatments. Water was restored after sampling at 74 DAP in 350DPI and 350DBS treatments, and at 75 DAP in 700DPI and 700DBS treatments.

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80 Table 4. 2. Sucrose phosphate synthase activities of rice plants grown at 350 and 700 L L 1 and in four different water managements. Plant samples were taken at noon eastern standard time from 100, 110 (stressed) and 127 (recovery) day old plants. L L 1 350 700 Note: Water Management FLD DPI DAN DBS FLD DPI DAN DBS l00DAP mol 41.6.8 42.2.9 40.6.9 41.2.5 44.8.6 44.9.3 42.3.7 43.6.8 P=0.0303 P=0.5696 P=0.9848 FLD: continuously flooded SPS (g Activities ll0DAP fresh weight) 38.6.4 39.8.4 15.0.0 25.4.7 45.8.3 46.5.4 15.5.1 26.9.5 P=0.0114 P=0.0001 P=0.1890 DPI: drought imposed during panicle initiation DAN: drought imposed during anthesis DBS: drought imposed during both stages DAP: days after planting WM: water management 127DAP 1 h 1 28.4.1 27. 7. 4 28.1.3 27.7.2 34.8.9 32.2.2 32.7.0 30.6.4 P=0.0044 P=0.5426 P=0.7804 Paddy water was removed at 93 DAP in 350DAN and 700DAN treatments, and at 96 DAP in 350DBS and 700DBS treatments. Water was restored after sampling at 110 DAF in 350DAN and 700DAN treatments, and at 111 DAP in 350DBS and 700DBS treatments.

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CHAPTER 5 COORDINATION OF CARBON METABOLISM ACTIVITIES This chapter briefly describes the relationship between carbohydrate levels and SPS activity from data cited in previous chapters 2, 3 and 4. Increasing CO 2 increased sucrose and starch concentration and also SPS activity. The increase in sucrose concentration and SPS activity was relatively great er compared to starch concentration throughout the season (Figures 5.1 and 5.2). For continuously-flooded plants, SPS activity, sucrose and starch concentration were initially high and decreased with increasing plant maturity. Starch concentration declined earlier compared to SPS activity and sucrose concentration (Figures 5.1) A considerable decrease in starch was observed during the end of growing season, while sucrose concentration and SPS activity was maintained moderately high. The decrease in sucrose and starch concentration in leaves during late plant development was due to onset of grain filling as the carbohydrate pool in rice leaves were increasingly mobilized and exported to the developing grain. Rice leaves appear to store more sucrose than starch. The doubled CO 2 treatments consistently had greater SPS activity, which was associated with and helped maintain relatively higher sucrose 81

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concentration in leaves. 82 Increased SPS activity could have originated from increased SPS protein or from a kinetically more active form in high-CO 2 leaves. Water deficit at panicle initiation (Figure 5.1) substantially decreased sucrose as well as starch concentration and also SPS activity. When water was witheld during panic le initiation, SPS activity, leaf sucrose and starch concentration declined concurrently from the onset of stress through the most stressed leaf phase After water restoration, sucrose concentration and SPS activity rapidly recovered, but starch concentration slowly recovered. After plants regained turgor there was slower recovery in leaf starch concentration for up to 7 days. Water deficit at anthesis caused considerable decrease in sucrose, starch, and SPS activity. Repeated stressed leaves were relatively higher in sucrose and starch concentration and SPS activity compared to water-stressed leaves at anthesis. Water withholding during anthesis phase decreased the plant's ability to recover from declining SPS activity, leaf sucrose and starch concentration was lower compared to that of plants exposed to water stress in the panicle initiation phase. It is possible to conclude that under water stress the higher sucrose concentration over starch concentration was due to the increases in the ratio of newly synthesized sucrose to starch and to the degradation of starch, together with the reduction in sucrose export out of the leaves The doubled

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83 100 C .g, -e350FLD r 700FLD C: .g 350DPI I ~. 700DPI \ 40 \ \ \ 8 20 E s 054 "' 64 74 84 94 104 114 124 Figure 5.1. Time course of sucrose and starch concentration and SPS activity under continuously-flooded and water-deficit at panicle initiation treatments.

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~100------------~ 80 + '---' --. \ .. .. 64 74 84 94 104 114 -ie-350DAN -700DAN ~350OBS -11700OBS 124 Figure 5.2. Time course of sucrose and starch concentration and SPS activity under water deficit at anthesis and at both phase treatments 84

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85 CO 2 concentration was able to delay the effect of water deficit for one day. Relationships of SPS and carbohydrate can also be evaluated via CO 2 enrichment effects on photosynthesis and soluble protein CO 2 enrichment increased Pn which would increase triose production for sucrose and starch formation. However elevated CO 2 also decreased soluble protein and presumably also Rubisco, which is a major constituent of soluble protein. The question is why is SPS activity increased despite a decrease in soluble protein. One possibility is that increasing Pn increased hexose formation which led to selective mRNA transcription to enhance synthesis of SPS protein or of an SPS acti vase protein that causes increase in SPS activity. Another possibility is that inorganic phosphate, P t in the cytosol will decrease under CO 2 -enrichment and higher triose-P production. Decreasing P 1 levels causes dephosphorylation of SPS which results in increasing SPS activity. This hypothesis assumes that the SPS assay determines only the kinetically active form of SPS. Increasing SPS activity would increase sucrose production. Therefore, rice leaves partition more assimilate to sucrose rather than starch. Relationships of SPS and carbohydrates under water deficit can likewise be evaluated in terms of drought effects on Pn and soluble protein. When the plants were subjected to drought stress by withholding water, the initial response of

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86 the plant is stomata! closure. This response will inhibit CO 2 diffusion into leaves which will decrease photosynthetic rate and result in decreased carbohydrate formation and deactivation of SPS ac t ivity (Vassey et al., 1991). The cause of decreased SPS activity under mild water deficit could be the reverse of events under elevated CO 2 i.e., increased P i availability may cause phosphorylation of SPS thus decreasing SPS activity or less hexose could lead to less transcription for synthesis of SPS protein. A third cause for decreased SPS activity is possible. Water deficit also causes photoinhibition and increases protein turnover as well as increases proline and glycine betaine production. That was evident in this study from decreased soluble protein under water deficit. This would lead to decrease in SPS protein, which in turn would decrease SPS act i vity. Photoinhibition also broke down chlorophyll, thus resulting in decreased ATP and NADPH formation and also decreased CO 2 assimilation. Elevated CO 2 promoted larger reduction in leaf stomata! conductance. As a result, the plant stands grown at elevated CO 2 utilized the available soil moisture more slowly than those grown under ambient CO 2 High-CO 2 plants also decreased transpiration rate more compared to ambient-CO 2 plants under water deficit. Therefore, plants grown in CO 2 -enriched environment were able to partly compensate for the effect of water stress and to extend the growing period

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CHAPTER 6 SUMMARY AND CONCLUSION The global atmospheric CO 2 concentration and regional rainfall intensities and frequencies are predicted to change during the next century. These anticipated changes in CO 2 and water availability could have adverse effects on physiology of rice, based on previous elevated-CO 2 studies on rice (Baker et al., 1988, 1996a, 1996b; Rowland-Bamford et al., 1990; Makino et al., 1994), bean (Vassey and Sharkey, 1989; Vassey et al., 1991) and spinach (Zrenner and Stitt, 1991). The objective of this study was to evaluate the effect of doubled CO 2 and specific water deficits on rice photosynthesis, soluble protein and chlorophyll concentration, carbohydrates, and SPS activity. This chapter briefly describes and summarizes the results of this study. Photosynthesis, Soluble Protein and Chlorophyll Elevated CO 2 concentration increased leaf photosynthesis 40% throughout the season. Water deficit initiated on the same date decreased photosynthetic rate of ambient CO 2 leaves significantly earlier (about 1 day) than the rate of elevated CO 2 leaves. For example, at day 74 during the panicle initiation water deficit, reduction in photosynthetic rate due 87

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88 to water deficit was 96% in low-CO 2 leaves and 53% in high CO 2 leaves during the panicle initiation phase. Leaf soluble protein of elevated-CO 2 leaves was significantly lower on most dates of the growing season. The greatest differences in leaf soluble protein between high-CO 2 and low-CO 2 plants was 18%. Withholding water during panicle initiation decreased leaf soluble protein concentration by 62% in low-CO 2 leaves and by 56% in doubled-CO 2 leaves. Effects of water deficit during anthesis on leaf soluble protein were significant. Anthesis drought stress decreased soluble protein concentration by 62% in ambient-CO 2 and 55% in elevated-CO 2 plants. The average increase in chlorophyll concentration for enriched-CO 2 over ambient-CO 2 plants was 13%. During water deficit, elevated-CO 2 leaves had significantly higher chlorophyll concentration than low-CO 2 leaves. Reduction in chlorophyll concentration during water deficit in the panicle initiation phase was 62% in ambient-CO 2 leaves and 48% in elevated-CO 2 leaves During anthesis phase, water deficit decreased chlorophyll 73% in low-CO 2 plants and 54% in high-CO 2 plants. Rice plants exposed to two water stress cycles had decreased chlorophyll concentration of 76% in low-CO 2 leaves and of 62% in high CO 2 leaves. Carbohydrates Elevated CO 2 concentration significantly increased sucrose concentration throughout the season by 9%. Water

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89 deficit during panicle initiation decreased sucrose concentration by 85% in ambient-CO 2 and 85% in elevated-CO 2 leaves. Effects of water management treatment during an thesis significantly decreased concentration of sucrose by 69% in ambient-CO 2 and 58% in doubled-CO 2 leaves. Leaf starch concentration was significantly increased by [CO 2 ] treatments from 58 through 74 DAP. Effects of [CO 2 ] treatment on concentration of starch declined with plant maturity. During panicle initiation, water deficit decreased leaf starch concentration by 93 % in low-CO 2 and 92% in high CO 2 leaves. Water deficit during anthesis also significantly decreased starch concentration. Leaf doubling fructose concentration increased by 36% [CO 2 ] from 54 through 127 DAP. Effects of treatments on fructose concentration were significant. Water deficit during panicle initiation significantly decreased fructose concentration by 90% in low-CO 2 and 84% in elevated CO2 leaves. When water was withheld during the an thesis phase, fructose concentration decreased by 80% in low-CO 2 leaves and 45% in high-CO 2 leaves. SPS Activity Elevated [CO 2 ] increased SPS activity throughout the season by 6 to 16%. Water stress treatment during the panicle initiation phase significantly decreased SPS activity by 72% in ambient-CO 2 and 58% in doubled-CO 2 plants. When water was

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90 withheld during the anthesis phase, SPS activity decreased by 61% in ambient-CO 2 and 66% in elevated-CO 2 plants. Coordination of Carbon Metabolism Activities Rice is clearly a sucrose-storing plant. Environments such as CO 2 enrichment that increased photosynthesis acted to increase SPS activity and sucrose concentration. By contrast, when water deficit occurred, it dramatically decreased leaf photosynthesis and concurrently decreased SPS activity and sucrose concentration. Water deficit effects also decreased starch and fructose concentration. Effects of water deficit on sucrose, starch, and fructose concentrations lasted as long as 7 days after water was restored. SPS activity, however, had fully recovered. Elevated CO 2 Decreases Effects of Water Deficit Elevated CO 2 can partly compensate for the effect of water deficit on Pn, soluble protein, chlorophyll, carbohydrate concentration, and SPS activity by maintaining activities for 1 to 2 day longer. Elevated CO 2 also stimulated more rapid recovery of Pn, soluble protein, chlorophyll, carbohydrate concentration, and SPS activity after relieving water stress effects.

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106 Zhang, H., and P. S. Nobel. 1996. Photosynthesis and carbohydrate partitioning for the C 3 desert shrub Encelia farinosa under current and doubled CO 2 concentra tons. Plant Physiol. 110:1361-1366. Ziska, L. H., w. Weerakoon, o. s. Namuco, and R. Pamplona 1996. The influence of Nitrogen on the elevated CO 2 response in field-grown rice. Aust. J. Plant Physiol. 23:45-32. Ziska, L. H., and A.H. Teramura. 1992a. of growth and photosynthesis in rice Plant Physiol. 99:473-481. CO 2 enhancement (Oryza sativa). Ziska, L. H., and A.H. Teramura. 1992b. Intraspecific variation in the response of rice (Oryza sativa) to increased CO 2 -photosynthetic, biomass and reproductive characteristics. Physiol. Plant. 84:269-276. zrenner, R., and M. Stitt. 1991. Comparison of the effect of rapidly and gradually developing water-stress on carbohydrate metabolism in spinach leaves. Plant Cell Environ. 14:939-946.

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BIOGRAPHICAL SKETCH Widodo was born July 1, 1958, in Semarang, Central Java Indonesia. He received his bachelor's degree in Agronomy in November 1983 from University of Gadjah Mada at Yogjakarta. Beginning in April 1984, he served as a lecturer with the University of Bengkulu. In October 1986 he received a scholarship from the Government of Indonesia, under the USAID program to study for his M.S. Before he left for the U.S.A., he married Pratiwi Widiastuti on October 19, 1986. After a year he studied at Western Kentucky University, his wife joined him. In October 22, 1988, their son, Okky Wenkyca Widodo was born. He completed his M.S. in April 1989. Upon arrival in Indonesia, he returned to the University of Bengkulu to teach undergraduate Agronomy classes. In June 20, 1990, they received a fourth member of their family with the birth of a daughter, Intan Puspita Widodo. In 1992 he received another a scholarship from the government of Indonesia, under the USAID program to study for a Ph.D. In January 1993 he joined the Agronomy Department of the University of Florida to work in crop physiology, under the supervision of Dr. Kenneth Jay Boote. Upon completion to the degree requirement, he will resume his position at the University of Bengkulu, Indonesia. 107

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I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of D~osop~ Kenneth J. BoAe. Chair Professor of Agronomy I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doct ~ of Philos y k::/ -0 / rge :i::. Bowes rofessor of Botany I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. --:h e d G 1/'L-Cu V. iu Associate Professor of Agronomy I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Do~ctor Philosophy. ~.1fa, eon H. Allen Jr. Professor of Agronomy I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. 11 J;r,J;/tL. RaondN. Gallaher Professor of Agronomy

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This dissertation was submitted to the Graduate Faculty of the College of Agriculture and to the Graduate School and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy. k December, 1996 ~Dea, College Agriculture Dean, Graduate School


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AA00064785_00001.pdf
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AA00064785_00001.mets
METS:structMap STRUCT1 physical
METS:div DMDID ADMID The ORDER 0 main
PDIV1 1 Main
PAGE1 Page i
METS:fptr FILEID
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STRUCT2 other
ODIV1
FILES1