Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations
2000 Carbohydrate regulation of leaf development: Investigating the role of source strength, carbon partitioning and hexokinase signaling in regulating leaf senescence Adam Christopher Miller Iowa State University
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Carbohydrate regulation of leaf development: Investigating the role of source strength,
carbon partitioning and hexokinase signaling in regulating leaf senescence
by
Adam Christopher Miller
A dissertation submitted to the graduate faculty
in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY
Major Plant Physiology
Major Professors: Steven R. Rodermel and Martin Spalding
Iowa State University
Ames, Iowa
2000
Copyright ® Adam Christopher Miller, 2000. All rights reserved. UMI Number. 9977346
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Graduate College
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This is to certify that the Doctoral dissertation of
Adam Christopher Miller has met the dissertation requirements of Iowa State University
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issor
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Forthe Gf^cfuate' College iii
TABLE OF CONTENTS
CHAPTER 1. GENERAL INTRODUCTION 1 Dissertation Organizatioii 1 Background Information and Literature Review 2 References 13
CHAPTER 2. ELEVATED CO, EFFECTS DURING LEAF ONTOGENY: A NEW PERSPECTIVE ON ACCUMATION 24 Abstract 24 Introduction 25 Materials and Methods 27 Results 30 Discussion 32 Acknowledgements 37 References 37
CHAPTER 3. LEAF DEVELOPMENT AND ACCLIMATION TO ELEVATED CO, 44 Introduction 44 Elevated CO, and Tobacco Leaf Development 47 A New Paradigm to Understand Acclimation SO Mechanism of the Temporal Shift 52 References S3
CHAPTER 4. CARBOHYDRATE REGULATION OF LEAF DEVELOPMENT: PROLONGATION OF LEAF SENESCENCE IN RUBISCO ANTISENSE MUTANTS OF TOBACCO 59 Abstract S9 Introduction 60 Materials and Methods 62 Results 64 Discussion 68 Acknowledgements 71 References 71
CHAPTER 5. CHANGE IN CARBON PARTITIONING DURING ARABIDOPSIS LEAF DEVELOPMENT 83 Abstract 83 Introduction 84 Materials and Methods 86 Results 89 Discussion 93 References 97 iv
CHAPTER 6. GENERATION OF TRANGENIC ARABIDOPSIS WITH ALTERED LEVELS OF HEXOKINASE (HXKl) ACTIVITY 113 Introduction 113 Materials and Methods 113 Results and Discussion 117 References 120
CHAPTER 7. GENERAL CONCLUSIONS AND FUTURE RESEARCH DIRECTIONS 127 Conclusions and Future Research 127 References 134
ACKNOWLEDGEMENTS 138 1
CHAPTER 1. GENERAL INTRODUCTION
Dissertation Oiigaiiization
This dissertation is organized into six chapters. Chapter 1 is an introduction to source strength and cart)ohydrate effects on plant systems. Specifically, this background information is divided into three main sections. The first section covers known carbohydrate effects on plant metabolic systems, particularly their effects on gene expression. The second section describes leaf development and factors involved in the senescence phase of development. The third section details the hexokinase sugar-signaling pathway in plants as a potential regulation step in carbohydrate effects on development. Chapters 2,4, and 5 are presented as independent journal papers; chapter 3 is a chapter in a book. Chapter 2 covers the experiments performed on tobacco plants under elevated COj conditions and how this affects the progression of leaf senescence. Chapter 3 summarizes these results and discusses their implications on our understanding of acclimation to high CO; environments. Chapter 4 covers the experiments performed on rbcS antisense mutants of tobacco and how decreased source strength effects on senescence compares to the increased source strength condition.
Chapter 5 covers the investigations of wild type Arabidopsis leaf development and how carbon partitioning changes over ontogeny and how these changes relate to hexokinase and invertase activities. Chapter 6 provides a description of the generation of transgenic
Arabidopsis plants with altered levels of hexokinase to be used in future analysis. Chapter 7 presents general conclusions and potential future projects. The last section contains the acknowledgements. 2
Background Informatioii and Literature Review
Carboiiydrates and Regulation of Gene Expression
The ultimate purpose of photosynthesis in higher plants is to fix CO2 and generate carbohydrates. After fixation, there are two main fates for this carbon: production of sucrose for export to other parts of the plant, or as starch for storage (Huber, 1989). These carbohydrates serve as the main source of chemical energy to power all of the cellular processes necessary to sustain life. However, mounting evidence has shown that carbohydrates have a much more complex role in plant metabolism besides simply as an energy supply. Sugars are now implicated as key regulatory molecules in several biochemical pathways. Abundance or insufficient levels of carbohydrates has been seen to either enhance or repress the expression of specific genes (reviewed in Koch, 1996; Graham,
1996). The specificity of action on gene expression suggests that this is a directed effect on sugar-modulated genes only, rather than a more general response to changes in energy supply status (Koch, 1996). Many of the major targets of carbohydrate regulation are genes involved in maintaining their own production and metabolism, consistent with a feedback regulation system (Koch, 1996; Hesse et al., 1995; Ko^mann et al., 1991; Krapp and Stitt,
1994). In general, plentiful amounts of sugars lead to an increase in genes responsible for storage and utilization of carbohydrates and a decrease in production genes. Conversely, sugar depletion enhances production genes and storage mobilization genes (Sheen, 1990;
Koch, 1996). Such modifications help the plant maintain an optimum balance between carbohydrate supply and demand. 3
Initially, outside of seed reserves, the "source" of all carbohydrates in the plant arises from photosynthetic activity in the chloroplasts of a "source" leaf. These carbohydrates are eventually converted into the sugar sucrose in the cytosol, and then exported out of the cell.
Sucrose is transported via the phloem to tissues that have an energetic requirement for these carbohydrates. Such tissues are also referred to as "sinks" for carbohydrates. Several observations have suggested that photosynthetic production of carbohydrates may be adjusted in response to the demand for these resources by the sinks. When sink demand is low, photosynthesis is down-regulated, and high demand results in increased photosynthesis
(Sonnewald and Willmitzer, 1992). The degree of demand by sinks would determine sucrose export out of the source leaf, and ultimately carbohydrate status of the cell. Therefore, low demand for photoassimilates would result in carbohydrate accumulation in source cells, leading to feedback down-regulation of photosynthetic gene expression to reduce production
(Stitt, 1991). A series of investigations have contributed support to this hypothesis.
Decreased sink demand was artificially created in these experiments by glucose feeding to spinach leaves (Krapp et al., 1991); cold-girdling spinach leaves to prevent export (Krapp and Stitt, 1995); and expressing a yeast apoplastic invertase in tobacco plants interrupting phloem loading (Sonnewald et al., 1991). In all cases, sugars were seen to accumulate in source leaves, and an inhibition of photosynthesis accompanied this change in sugar partitioning. In some cases, the effects of sugar accumulation were not apparent until the leaf had reached a certain developmental stage (Sonnewald et al., 1991). This suggests that sugar modulation of photosynthesis may interact with other developmental factors. Importantly, recent studies have also implied a more complicated mode of regulation of photosynthesis besides purely down-regulating expression of photosynthetic genes. Mature spinach leaves 4 exogenously supplied glucose demonstrated not only an inhibition of pbotosynthetic protein synthesis, but also an increase in degradation as well (Kilb et al., 1996). Overall, changes in sugar status are shown to affect photosynthesis, demonstrating that carbohydrate export can play a role in controlling production rate.
However, the influence of sugars over resource allocation and use extends beyond self-regulation solely. The list of gene classes found to be responsive to changes in carbohydrates is extensive and varied. Reports include carbohydrate-induced changes in genes involved in pigment metabolism and defense responses (Kim et al., 1991), as well as mitochondrial genes (Felitti and Gonzalez, 1998). Genes responsible for macronutrient metabolism have also exhibited changes to carbohydrate levels. For example, nitrate reductase, a key enzyme in nitrogen uptake and processing, has exhibited enhanced expression in Arabidopsis in response to sugars (Cheng et al., 1992). Exogenous sucrose has also been observed to repress expression of asparagine synthetase in Arabidopsis (Lam et al.,
1994). Such results imply a role for carbohydrate regulation that encompasses coordinating the entire nutrient status of the plant. Ultimately, this regulation may extend to inducing changes in whole plant morphology and development, especially alterations concerning allocation of resources. For instance, tuber induction in potato was observed to be affected by sucrose application (Simko, 1994).
The site of action of effects on gene expression appears to be focused primarily on transcription (Sheen, 1990; Krapp et al., 1993; Morita et al., 1998). Investigations into the patatin promoter in potato, which is inducible by sugars, revealed sequence elements conserved in other sugar-responsive in sweet potato, suggesting there may be a common regulatory mechanism (Grierson et al., 1994). However, similar examinations of 5 photosynthetic gene promoters revealed no clear consensus regulatory sequence (Sheen,
1990). This suggests that carbohydrate effects on gene expression may be elicited via several different mechanisms.
In summary, there is much data to support the notion that carbohydrates play a role in regulating photosynthesis, and that this control is exerted on the gene expression level.
These conclusions are consistent with the prevailing hypothesis that sink demand for carbohydrates is a key determinant of photosynthetic rate. There is also evidence however, that carbohydrates have a more substantial role in regulating expression of genes involved in other forms of nutrient utilization. Also, the changes in photosynthesis display similarities to changes observed during leaf development, rather than simply representing a temporary down-regulation of expression (Kilb et al., 1996).
Leaf Development and Senescence
Put simply, leaf development refers to the progression of change that occurs throughout ontogeny. The leaf is a complex organ, with many different components and processes. Proper growth and differentiation into a functional organ requires a highly ordered series of events to take place, including initiation, cellular expansion and division, and specific tissue formation (reviewed in Van Lijsbettens and Clarke, 1996).
Implementation of such a systematic program is accomplished through genetic control
(reviewed in Van Lijsbettens and Clarke, 1998; Bnitnell and Langdale, 1996). Several investigations have taken advantage of mutant analyses to identify numerous genes responsiuie for maintaining normal developmental characteristics, such as leaf initiation
(Long et al., 1996), cell division and elongation (Talbeit et al., 1995; Kim et al., 1996), and 6 cell differentiation (Yang and Sack, 1995; HUlskamp et al., 1994; Pzremeck et al., 1996).
However, this genetic control is not autonomous, and relies on the transmission and perception of certain signals to coordinate their expression (reviewed in Brutnell and
Langdale, 1998).
Leaf development does not refer solely to observed changes in cellular and organ morphology. There are many changes throughout ontogeny that occur at tne biochemical level. One of the most studied is the change in photosynthetic capacity that occurs with leaf development. In general, the pattern of change in photosynthetic capacity of a dicot leaf can be divided into two major stages (reviewed in Gepstein, 1988). The first stage is one of increasing photosynthetic rates over time. This stage coincides with rapid cell division and leaf expansion. As photosynthetic rates increase and cell division slows down around full expansion, the leaf converts from a net importer of carbohydrates (sink) to a net exporter
(source) (Huber, 1969). The second stage exhibits a gradually decline in photosynthetic rates over time. This phase is also referred to as "senescence". Generally, the term senescence deals with developmental processes that ultimately lead to death. In essence, senescence is an ongoing phenomenon, such as in the formation of xylem tracheids (Matile, 1992).
However, leaf senescence is a specific developmental process involving a decline and remobilization of cellular components and eventually leading to leaf death and abscission
(reviewed in Brady, 1968). The major events associated with this phase, besides the change in photosynthetic rates, are the declines in chlorophyll and protein levels (Matile, 1992;
Lx)hman et al., 1994). In fact, the change in photosynthetic rate has been shown to be due primarily to changes in Rubisco activity (Jiang et al., 1993). Existing foliar proteins and chlorophyll are broken down to facilitate mobilization to other plant parts, with the majority 7
of the degradation occurring in the chloroplasts (Matile, 1992; Gan and Amasino, 1997).
However, this degradation requires the presence of specific proteins to carry out the
proteolysis and remobilization. Therefore, the senescence stage of development requires
synthesis of proteins as well as breakdown. There can be no broad based, nonspecific
degradation of cellular components. It has also been demonstrated that not only is
degradation targeted to specific components, but transcript levels also change in a specific
manner, and this phenomenon is applicable to many different species. Levels of
photosynthetic mRNAs are seen to decline in senescent tissue (Hensel et al., 1993; Jiang et
al., 1993). Conversely, genes responsible for degradation and remobilization of resources
exhibit increased mRNA levels (Hensel et al., 1993; Kamachi et al., 1992; Taylor et al.,
1993; Drake et al., 19%). Inhibitor studies that blocked RNA synthesis also showed that leaf
senescence could not proceed without changes in gene expression. These results suggest that
senescence is under genetic control (reviewed in Nood6n, 1988; Buchanan-Wollaston, 1997).
Similar to other leaf developmental programs, this genetic control is subject to modulation
via the perception and transmission of internal and external factors.
Several factors have been demonstrated to have an effect on the progression of senescence. The major factors identified include plant hormones, light, nutrient status, environmental stresses (i.e., temperature, drought, wounding, etc), and developmental age
(Gan and Amasino, 1997). For simplicity sake, we will only consider hormones, light and sugar effects here, but it is important to remember that other influences exist All of the major hormones may have some role during the senescence phase, but only cytokinins and ethylene have been demonstrated to exert regulatory control (Smart, 1994). The presence of cytokinins in a leaf has been shown to retard senescence in many different plant species 8
(Nooddn et al., 1990). Senescence delay was observed both with exogenous application of
cytokinins, and overproduction via genetic manipulation (van Staden et al., 1988; Gan and
Amasino, 1995). This regulation by cytokinins appears to be at the level of transcription
through a signaling pathway (Buchanan-Wollaston, 1997). Conversely, ethylene has been
associated with the initiation of the senescence process (McGlasson et al., 1975). Exogenous
application of ethylene has been shown to promote chlorophyll and protein loss in some
cases, and application of ethylene biosynthesis inhibitors retarded these similar
characteristics of leaf senescence (Grbic and Bleeker, 1995). It was also demonstrated that
ethylene production and responsiveness was modulated at the level of gene expression (John
et al., 1995; Grbic and Bleeker, 1995). However, these responses were not observed in
young seedlings, suggesting that ethylene effects are only apparent when the leaf is already
"prepared to senesce". Therefore, it has been proposed that ethylene may increase the
sensitivity of the interaction of other "age-related" factors, rather than having a true
regulatory role (Buchanan-Wollaston, 1997). Recently, the plant growth regulator methyl
jasmonate has also been implicated as having a role in leaf senescence (Chen and Kao, 1996;
Beltrano et al., 1998). However, only its participation has been observed, and further investigation is necessary to elucidate its true role. The effects of light on leaf senescence are at best, complex. In some instances, light may act to promote senescence, and in other instances it may be darkness (Biswal and Biswal, 1964). There is evidence that phytochrome signaling may sense low light levels, triggering senescence (Rousseaux et al., 1996). Other experiments have shown that high quantities of light achieved through intensity changes and long day growth periods may also accelerate senescence (Nood^n et al., 1996). Although not tested, this light effect may be attributed to changes in photosynthate production. 9
It has been suggested that the initial decline in photosynthetic rate may represent the sig:ial for senescence induction. Sucrose depletion was found to be associated with foliar senescence of asparagus (King et al., 1995). However, other studies contradict this hypothesis. Some experiments showed that gene expression changes indicative of senescence occurred before any observable losses in photosynthetic rate or chlorophyll amount (Kilb et al., 1996). Carbohydrate status of the leaf has also been shown to have contrary effects on senescence (Crafts-Brandner et al., 1984; Frdhlich and Feller, 1991). 1 have already discussed the effects of carbohydrates on photosynthesis via feedback sink regulation. Therefore, it is reasonable to hypothesize that photosynthetic decline during senescence is related to carbohydrate accumulation in a similar fashion. Since several inputs regulating senescence have been identified, carbohydrates must interact with these factors somehow. For example, one study determined that sugar accumulation can block the inhibitory effect of cytokinins on senescence, but that glucose repression is affected by light levels (Wingler et al., 1998). In fact, sugars may represent the "age-related" factor necessary for ethylene sensitivity. Recent investigations have suggested that expression of genes necessary for ethylene biosynthesis may be up regulated by sugars (Sonnewald et al., 1995).
Current studies are also revealing an interaction between sucrose and jasmonic acid on plant development (Kovac and Ravnikar, 1998). Figure 1 illustrates the potential model for carbohydrate regulation of leaf development Therefore, sugar-mediated developmental effects would require some sort of sensing mechanism to elicit changes in gene expression. 10
Hexokinase and Sugar Signaling
Carbohydrate signal transduction pathways have been identified in numerous organisms. The most well characterized of these systems is the glucose repression pathway in Saccharomyces cerevisiae (reviewed in Gancedo, 1992; Trumbly, 1992). There are several components to this pathway, including a protein phosphatase; GLXT?, a regulatory subunit; REGl, a repressor complex; consisting of CYC8 and TUPl; and a DNA binding protein; MlGl (reviewed in Trumbly, 1992). However, the initial sensor of glucose levels is thought to be hexokinase Pll, encoded by the HXK2 gene (reviewed in Trumbly, 1992;
Smeekens and Rook, 1997). This hexokinase, along with two other proteins, HXKl and
GLK, is responsible for phosphorylating glucose. Glucose is the preferred energy source in yeast, and this step is required for further metabolism and utilization of this carbon source
(reviewed in C^denas et al., 1998). In the presence of glucose, genes responsible for the metabolism of alternate carbon sources such as sucrose and galactose are down-regulated, perhaps as a means of conserving resources (Smeekens and Rook, 1997). This change in gene expression requires phosphorylation by hexokinase to occur (Rose et al., 1991).
Although glucokinase phosphorylation results in the same end-product, no repression is observed (Ma and Botstein, 1986). The hxkl protein can initiate sugar repression, but only represents 10% of the generated signal, with hxk2 responsible for the other 90% (Ma and
Botstein, 1966). There is still debate as to whether or not catalytic activity and signaling are correlated (Entian and Frdlich, 1984; Ma et al., 1969). It is also unknown how the actual signal is generated by this activity, however it is clear that hexokinase phosphorylation is necessary for sugar sensing in yeast. 11
There is mounting evidence higher plants share components of the sugar signaling pathways identified in yeast. Plant homologues are being discovered in several species, such as the presence of SNFl-like protein kinases. The SNFl gene is involved in the glucose derepression pathway in yeast, and similar kinases have been observed in potato (Man et al.,
1997), tobacco (Muranaka et al., 1994), and rye (Alderson et al., 1991). Even more convincing, is the discovery cf a SNF4-iike protein in Phaseolus (Abe et al., 1995). Unlike the SNFl protein, which has various roles in yeast, the SNF4 protein appears to be very specific for carbon signaling (reviewed in Gancedo, 1992). Interaction between SNFl and
SNF4 has been demonstrated to be regulated by glucose (Jiang and Carlson, 1996).
Recently, the aforementioned Phaseolus gene has been shown to complement the snf4 mutation in yeast (data not published). Of course, one would expect hexokinase to be evolutionarily conserved, due to their importance in metabolism. However, hexokinase's role as a sugar sensor in plants is not as intuitive, and is just beginning to be elucidated.
Several recent studies have served to provide very strong evidence that hexokinase can act as a sugar sensor in higher plants. They have also suggested that the feedback inhibition of photosynthesis discussed earlier may result from carbohydrate signaling via this pathway (Jang and Sheen, 1994). These experiments examined expression of chloramphenicol acetyltransferase (C!AT) reporter genes fused with several different photosynthetic promoters in a maize protoplast transient expression system. In all cases,
CAT expression was greatly decreased with the supply of sugars that can be phosphorylated by hexokinase (HXK) (Jang and Sheen, 1994). A glucose analog that is not a substrate for hexokinase, 3-0-methylglucose, did not alter CAT expression, demonstrating that phosphorylation by HXK is necessary and the observed results were not due to osmotic 12 effects (Jang and Sheen, 1994). Further metabolism of phosphorylated hexoses was also not necessary for repression. 2-deoxyglucose, an analog that can be phosphorylated by hexokinase but metabolized further, still displayed repression of CAT activity (Jang and
Sheen, 1994). Excess phosphate and ATP provided were not able to alleviate hexokinase- mediated repression (Jang and Sheen, 1994), showing that depletion of these components were not responsible for repression as has been hypothesized (Huber, 1969; Loughman et al.,
1989; Brauer and Stitt, 1990). Another set of experiments examined transgenic Arabidopsis thaliana plants with either increased or decreased levels of hexokinase (Jang et al., 1997).
Mutant seedlings overexpressing hexokinase demonstrated hypersensitivity to high glucose growth conditions, while antisense mutants with low hexokinase levels showed hyposensitivity to high sugars (Jang et al., 1997). It should be noted that two hexokinase genes were cloned, and that transcripts for both were reduced in antisense mutants containing constructs specific for only one of them (Jang et al., 1997). Changes observed in gene expression of cab and rbcS transcripts also reflected the differences in sensitivity to glucose due to altered hexokinase levels. Overexpression mutants had a greater degree of glucose- mediated repression compared to wild type plants, while antisense mutants maintained higher levels of expression (Jang et al., 1997). These results provide excellent evidence that hexokinase is involved in sugar signaling in vivo. Importantly, hexokinase was demonstrated to have an effect on a developmental level, as well as a whole organismal level.
Free hexoses, primarily glucose and fructose, are the known substrates for hexokinase activity. However, as illustrated in Figure 2, photosynthetic production of sucrose and/or starch does not generate free hexoses. In order for hexokinase to serve as a sensor of carbohydrate production, these substrates must be available. One very good candidate for 13 potential supply of free hexoses for hexokinase signaling is through the action of acid invertase (Koch et al., 1996; Cheng et al., 1996). The irreversible hydrolysis of sucrose by invertase generates glucose and fructose, which are both suitable substrates for phosphorylation by hexokinase. This is also illustrated by Figure 2. It has also been shown that in some species, substantial acid invertase activity can be maintained in the leaf after maturity to a source tissue (Huber 1969). Since free hexoses are not seen to accumulate due to this activity, it is reasonable to theorize that they are phosphorylated and returned to the sucrose biosynthesis pathway (Huber 1969). Sucrose would be expected to accumulate when export to sinks is limited, and could therefore partition to this pathway and generate potential signal. This is still consistent with the sink limitation hypothesis discussed earlier. Another sucrose cleaving enzyme, sucrose synthase, could also represent a potential signal generator.
However, this enzyme produces fructose and UDP-glucose as its end products; half the potential signal of its invertase counterpart (Koch et al., 1996). Also, expression of this enzyme appears limited mainly to developing sinks such as reproductive structures and storage organs (Koch et al., 1996). In order to test the role of invertase in sugar signaling, our lab is currently in the process of conducting expression experiments.
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SIGNAL TRANSDUCTION LEAF DEVELOPMENT (SENESCENCE)
Hcrmonai (cytokinins, athylana, ate)
Envlranmantal Straas ;taar (drought, tamparatura, ate) cktoraflMt MM OttMrsanMconct Ught OtNr m«tabolic «nd •vants physiological UtXon CsrMiiirata PrMncttaa (isiHC* atr«ii|tlil
Figure 1. Potential model for carbohydrate regulation of leaf development
Staixh^ucrose Biosynthesis in a Source Leaf Cell
CytiMol
Hgure 2. Schematic representation of carbohydrate flow in a source leaf cell. 24
CHAPTER 2. ELEVATED COj EFFECTS DURING LEAF
ONTOGENY: A NEW PERSPECTIVE ON ACCLIMATION
A paper published in Plant Physiology 1 IS: 119S-2000 (1997)
Adam Millei*'', Chiu-Ho Tsai', Dean Hemphill*'^ Matt Endres, Steve Rodermel*-'', and Martin
Spalding'^''
Abstract
For many plants, growth in elevated CO} leads to reduced rates of photosynthesis.
To examine the role that leaf ontogeny plays in the acclimation response, we monitored photosynthesis and some related parameters at short intervals throughout the ontogenetic development of tobacco (Nicotiana tahacum L.) leaves under ambient (350 /iL L'') and high (950 )iL L'') CO, conditions. The pattern of photosynthetic rate over time was similar between the two treatments and consistent with the expected pattern for a typical dicot leaf. However, the photosynthesis pattern in high-C02-grown tobacco was shifted temporally to an earlier maximum and subsequent senescent decline. Ribulose-1,5- bisphosphate carboxylase/oxygenase activity appeared to be the nuun factor regulating photosynthetic rates in both treatments. Therefore, we propose a new model for interpreting the acclimation response. Lowered photosynthetic rates observed during acclimation appear to be the result of a shift in the timing of the normal photosynthetic
' Department of Botany, Iowa State University, Ames, Iowa 50011
" Interdepartmental Plant Physiology Major, Iowa State UniversiQr, Ames, Iowa 5(X)11 25 stages of leaf ontogeny to an earlier onset of the natural decline in photosynthetic rates associated with senescence.
Introduction
Atmospheric CO2 levels are expected to almost double within the next century if current emissions are not reduced, so an understanding of how higher plants cope with such a dramatic environmental change becomes a question of vital importance (Bazzaz, 1990;
Bowes, 1993). One of the more intriguing responses of plants to elevated CO, is the phenomenon of acclimation (Stitt, 1991; Bowes, 1993). In many Q plants under normal growth conditions, photosynthetic rates are limited by the carboxylation rate of Rubisco, the key regulatory enzyme of photosynthetic carbon assimilation (Sage, 1994; Gutteridge and
Gatenby, 1995). Increasing CO, availability should increase the carboxylation rate of
Rubisco, and, commensurate with the expectation from Rubisco kinetics, plants grown under elevated CO, conditions initially respond with an increase in photosynthetic rate. However, this assimilation increase is lost in many plants after several days of exposure to elevated-
CO; conditions, and photosynthetic rates become only marginally higher or even lower than those observed prior to exposure to the increased CO;. The decrease in photosynthesis is particularly apparent when photosynthetic rates are measured under the same CO2 concentration, since the photosynthetic rates of C02-grown plants then are lower than those of ambient COj-grown plants (Sage, 1994). It is this apparent down-regulation of photosynthetic activity that is termed acclimation (Stitt, 1991).
The mechanisms underlying acclimation remain unclear. According to the sink limitation hypothesis, the inability of a plant to develop new sink tissue is a major factor in 26 determining acclimation sensitivity (Stitt, 1991). If sink production is insufficient to consume the higher quantities of photosynthate produced under high CO, conditions, photosynthetic rates are down-regulated to match the demand of the sink for carbohydrates.
One proposed mechanism for this sink regulation is that a feedback signal to down-regulate photosynthetic gene expression is generated in the source leaves by carbohydrate (sugar) accumulation, by other metabolites generated in respor«e to sugar accumulation, or by flux through specific metabolic pathways (Jang and Sheen, 1994; Koch, 1996). The theory of sink limitation has been well supported by many past experiments, including COj-enrichment studies in tobacco (Nicotiam tabacum) (Sicher et al., 1994).
Most studies of the acclimation response have concentrated on mature, fully- expanded leaves. This type of approach overlooks the normal pattern of changing leaf photosynthetic rates over extended time periods. During their ontogeny, dicot leaves undergo two distinct photosynthetic phases: a phase of increasing photosynthetic rates, which is normally correlated with leaf expansion, and a prolonged senescence phase of declining photosynthetic rates, with a transient peak of maximal photosynthetic rates between the two phases (Gepstein, 1988). We began to question whether leaf age may play a role in the interaction between source/sink balance and acclimation when studies with a Rubisco small- subunit antisense mutant of tobacco suggested that source strength may influence the onset and duration of these photosynthetic phases (Jiang and Rodermel, 1995).
Consistent with this possibility, several studies have suggested that there may be interactions between leaf ontogeny and the severity of the acclimation response (Besford et al., 1990; Xu et al., 1994; Nie et al., 1995; Pearson and Brooks, 1995; Van Oosten and
Besford, 1995; Van Oosten et al., 1995). In this report, we examine the photosynthetic 27 response of tobacco leaves to enriched CO, as a function of leaf age, beginning with early leaf expansion and continuing throughout their ontogeny. Our results suggest that the classical interpretation of acclimation may require revision, at least in the case of tobacco.
Matcriab and Methods
Plane Material and Growth Conditions
Isogenic lines of tobacco (Nicotiana tabacum SRI) seeds were germinated in 10-cm pots in the greenhouse. After germination, the pots were moved into an interim growth chamber (15 hour photoperiods at approximately 300 /imol photons m'^ s-^ 2S*'020°C, and
^75% RH), employing both incandescent and fluorescent bulbs as light sources. Pots were watered daily with an ammonium nitrate supplement, and seedlings were transplanted to 21- cm pots when five true leaves were clearly visible (approximately 1 month after planting).
Osmocote slow-reiease fertilizer was added to the soil after transplanting, and the plants were watered daily to the drip point.
Plants were moved into controlled-CO: experimental chambers (Model El 5,
Conviron, Asheville, NC) when leaf 10 (counting up from the base) reached 1 cm in length.
Day-one status was assigned to leaf 10 when it had reached a sufficient size for analysis (3 cm in width by 5 cm in length), approximately 5 d after placement into the chamber. The experimental chambers provided the same growth conditions as the interim chambers, except that CO3 concentrations were set at either zero injection for ambient CO, levels
(approximately 350 fiL L*') or 950 ftL for high CO; conditions. 28
Photosynthesis and Transpiration Measurements
Measurements of CER (The photosynthetic rate), Cs and [C]; were performed using a portable, closed-chamber photosynthesis unit (model 6200. Li-Cor, Lincoln, NE) as previously described (Jiang et al., 1999). CERs were measured 1 hour before the end of the light period in ambient COj concentrations (approximately 350 /iL L'O for both sets of plants. Plants exposed to 950 ftL L ' CO2 were moved to ambient-CO: chambers and allowed to adjust to these conditions for 10 min prior to taking measurements. Three readings were taken at 10-s intervals and averaged for a fmal value. The measurements were rechecked 10 minutes later to confirm the initial readings. Two to three individual plants were measured separately for each leaf age, and because they were sampled destructively, each plant was used for only one set of CER measurements. Immediately after the photosynthesis measurements, 0.9-cm leaf discs were punched out from the middle third of the leaf (where CER measurements had been taken), snap-frozen in liquid Nj and stored at -
80°C for future analysis.
Samples for determination of chlorophyll content (Amon, 1949) were prepared from two leaf discs ground to a fme powder in liquid Nj, and then extracted with 80% acetone.
Rnbisco Activity Assays
All Rubisco activity assays were performed in a cold room (4''C) using procedures similar to those described by Sicher et al. (1994). Two leaf discs were ground in liquid N,, and then thawed by adding 0.5 ml of extraction buffer [lOOmM 4-(2-Hydroxyethyl)-l-
Piperazine Propane Sulfonic Acid/KOH to pH 7.1,2mM EDTA, 1% (w:v) PVP, 20mM
MgSOa, 2(hnM KH2PO4,5mM e-aminocaproic acid, 2mM benzamidine, 20mM ascorbate. 29
5mM DTT, and lOO^iM PMSF]. After centrifugation for 10 s in a microcentrifuge, the soluble fraction was removed and assayed immediately for initial and total Rubisco activities.
For initial activity, assays were initiated within 40 s after the addition of the extraction buffer
by the injection of 20 jiL of extract into plastic mini-scintillation vials (in triplicate) containing 480 /iL of reaction buffer (lOOmM Tris/HG to pH 8.1,20niM MgCl2,0.4mM
RuBP, and lOmM •''C-Iabeled NaHCQa). The vials were then capped with serum stoppers and incubated at 25''C for 60 s. The reactions were terminated by the addition of 0.1 ml of formic acid, uncapped, and placed in a fume hood oveniighL The incorporation of into acid-stable products was determined by liquid-scintillation spectroscopy.
To determine the total activity the soluble Rubisco fraction was incubated in the reaction buffer (minus RuBP) for 5 min at 2S*'C to attain complete activation. RuBP was then injected into the vial to begin the reaction. All other procedures were carried out as in the initial activity assay.
Protein Content
Procedures for the isolation of soluble leaf proteins and SDS-PAGE electrophoresis have been described previously (Rodermel et al., 1968). Twelve-percent discontinuous polyacrylamide gels were stained with Coomassie blue G-250 as described by Neuhoff et al.
(1968), and dried between two sheets of gel drying film. The relative abundance of the
Rubisco large and small subunit proteins in each gel lane was then determined by densitometry, also as previously described (Rodermel et al., 1968). 30
Results
As illustrated in Figure 1A, growth in elevated CO, did not significantly affect either the rate of expansion or the final size of leaf 10 relative to the ambient control. However, the final dry weight of the high COj-grown leaf was 40% greater than that of the ambient CO2- grown control (data not shown), indicating a lower specific leaf area (centimeter/gram) for the high COi-grown leaf. Figure IB illustrates how the leaf photosynthetic rate (CER) measured in ambient COj changed with leaf age in plants grown under ambient or elevated
COj. Under ambient-CO, conditions, CER increased to a maximum at d 12, about 2 d after full leaf expansion, followed by a steady decline in photosynthetic rate, as is typical of dicot leaves (for review, see Gepstein, 1988). Leaves of plants grown under high CO, attained a
CER maximum similar to that in leaves from ambient-COj-grown plants, and they subsequently underwent a comparable rate of photosynthetic decline. However, relative to their ambient-COj-grown counterparts, the high-COj-grown leaves reached their photosynthetic maximum and initiated the photosynthetic decline at a significantly earlier leaf age (d 4 versus d 12) and degree of leaf expansion (50% versus 100%). This suggests that the initiation of the photosynthetic rate decline was shifted temporally in high-CO, conditions.
To explore the possible involvement of van'ous parameters in controlling the photosynthetic rates in Hgure IB, we first examined Cs and [C],. Although Cs changed with leaf age (Fig. IC), there appeared to be little difference between the two treatments. The change in [C]; with leaf age, which is calculated from CER and Cs values, is shown in
Figure ID. The higher [C]; values observed in high-C02-grown leaves after d 6 resulted from similar Cs values for both types of leaves coupled with lower photosynthetic rates in leaves 31 exposed to high CO;- Stomatal conductance can limit photosynthesis by restricting the
availability of CO,, but the higher [C]j values of high-COj-grown leaves indicate that the lower CERs observed after d 6 did not result from reduced CO3 availability. The Cs and [C]i results together suggest that stomatal effects were not a major component regulating photosynthetic rates in the two treatments.
To evaluate the potential role of light harvesting in regulating CERs in both sets of plants, we measured leaf chlorophyll concentrations (Fig. 2A) and found the pattern of change to be similar to that of CER. The rate of chlorophyll loss associated with the photosynthetic decline was similar for each growth condition, but the onset of loss of chlorophyll appears to have been shifted temporally in the high-COj-grown leaves, occurring at a much eariier leaf age than in leaves under ambient-CO; conditions. These data suggest that changes in light-harvesting capacity may have played a role in the regulation of photosynthetic rates in leaves of the two treatment types, or may have been controlled by another factor in parallel with CER.
Since photosynthetic rates often are limited primarily by the ability of Rubisco to fix
CO2 (Besford et al. 1990, Jiang et al., 1993; Stitt and Schulze, 1994; Jiang and Rodermel,
1995), Rubisco activity is a likely candidate for controlling the observed CER patterns. The initial activity of Rubisco, which is an estimate of in vivo-activated Rubisco, is shown in
Rgure 2B as a function of leaf age. Leaves grown in ambient-CO; conditions exhibited very high Rubisco activity eariy in leaf ontogeny, when the leaves were still expanding. After a significant drop between d 6 and 10, activities increased again to a peak coincident with the peak in CER at d 12 (Fig. IB). Initial Rubisco activity levels then decreased with increasing leaf age. Leaves grown in elevated COj had much lower levels of Rubisco initial activity 32 throughout leaf ontogeny. The highest Rubisco initial activity in high-CO^-grown leaves was observed at d 4, corresponding to the peak in CER.
Overall, these data show that the pattern of initial Rubisco activity is similar to that of
CER for both growth conditions, with the exception of d 4 and 6 in ambient-COj-grown leaves. This suggests that Rubisco activity may play a role in controlling CER, especially later in leaf ontogeny. These data also indicate that there was a temporal shift to an earlier loss of initial Rubisco activity in high-COj-grown leaves.
The total activity of Rubisco, which is a measure of the total amount of Rubisco that can potentially be activated in the leaf at the time of harvest, is shown as a function of leaf age in Figure 2C. The profiles of change in total activity in both sets of plants parallel the patterns of change in initial activity in Figure 2B, indicating that the in vivo activity of
Rubisco appeared to be directly correlated with the total amount of activatable Rubisco in the leaves. The relative amounts of Rubisco large subunit protein per unit leaf area measured by densitometry of SDS-PAGE gels were consistent with the total activity measurements (data not shown), confirming that less Rubisco protein was present in high-CO^-grown plants at comparable leaf ages. Taken together, these data demonstrate that there was a temporal shift to an earlier decline in Rubisco under high-COj conditions.
DiscosskHi
Photosynthesis over the life of a leaf can be divided into two distinct phases: a phase of increasing rates to a maximum, followed by a senescence phase of decreasing rates
(Gepstein, 1968). Both ambient- and high-C02-grown plants exhibited this general pattern of photosynthesis during leaf ontogeny, but in high-COj-grown plants there was a temporal shift 33 to an earlier transition from the first phase of increasing photosynthesis to the senescence phase of declining photosynthetic activity. This transition occurred on d 4 in high-CO,- grown leaves, 8 d before the transition occurred in ambient-COs-grown leaves. Therefore, although the maximum photosynthetic rates of the two were similar, high-COj-grown leaves entered the normal stage of photosynthetic decline several days before their ambient-COj- grown counterparts. The insensitivity of leaf expansion to growth in elevated COj demonstrates that the shift in timing of the photosynthetic decline cannot be explained simply by faster growth in elevated COj. Therefore, these observations indicate that acclimation, at least in tobacco, may represent an alteration of the timing of the normal leaf photosynthetic stages, rather than a direct photosynthetic down-regulation to another physiological state.
These two alternatives are illustrated in Figure 3. The dashed line represents the CER pattern expected over the life of a leaf if acclimation was simply a down-regulation of photosynthesis, in which case photosynthetic rates would decrease in magnitude, with no alteration in the timing of the normal photosynthetic phases. The temporal shift model suggests that the decreased photosynthetic rate is achieved by a shift in the timing rather than the magnitude of the phases of photosynthesis. Therefore, lower photosynthetic rates after prolonged exposure to high CO, (i.e., late in leaf ontogeny) would result from the natural ontogenetic decline of photosynthesis shifted temporally to an earlier onset, (Fig. 3, inner bold arrow). The Rubisco activity, Rubisco protein and chlorophyll data provide further support for this hypothesis, because loss of these components also appears to be shifted to an earlier leaf age in high-COj-grown leaves.
The correspondence between Rubisco initial activity and CER suggests that the changes in CER may be controlled largely by Rubisco activity. However, because the high 34
Rubisco activities observed for d 4 and 6 under ambient-COj-growth conditions are inconsistent with the observed CER, clearly Rubisco was not limiting photosynthesis on these days. Whatever the explanation for these high Rubisco activities, it is not clear why such a pattern was not observed in high-C02-grown leaves as well, unless the large peak in
Rubisco was shifted to a leaf age earlier than those we were able to measure. Even though leaf size limited our ability to measure CER during this time, it is apparent from the Rubisco data that some substantial changes occurred in the high-COj-grown leaves during the 5 to 7 d prior to our first measurements, which is consistent with a temporal shift model.
Earlier investigations by Besford et al. (1990) of the effects of CO, enrichment on photosynthesis and Rubisco during tomato leaf ontogeny resulted in observations somewhat similar to those reported here for tobacco. In those studies a similar peak CER for ambient- and high-COo-grown plants (measured at ambient CO3) was reported, both in magnitude and timing, but there was a more rapid decline in CER in the tomatoes grown in elevated CO2.
The authors concluded that the ontogenetic decline in photosynthesis was accelerated by the high-CO: treatment, a conclusion that would not be entirely consistent with either a temporal shift model or a surict photosynthesis down-regulation model. However, their data also show an earlier peak of Rubisco activity and a decline in Rubisco activity in high-COj-grown plants, which began earlier but was roughly parallel to that of the ambient-COj-grown plants.
Therefore, as would be expected based on the temporal shift model, the decline in Rubisco activity appeared to be shifted temporally rather than accelerated in rate.
Any conclusions about acclimation must consider that the response to elevated COj likely will be affected by the experimental conditions and the species investigated.
Therefore, it is difficult to evaluate the significance of the inconsistencies between the 35 curreot studies with tobacco and those of Besford et al. (1990) with tomato, especially since the source/sink relations of the two species are so different. It is not clear, for example, whether the differences between the obsen/ations in the two studies regarding the effect of elevated CO, on the pattern of CER as a function of leaf ontogeny reflect differences in experimental conditions, sink/source differences or species differences unrelated to sink/source balance. Because of this, we cannot be certain whether the temporal shift of the normal ontogenetic photosynthesis pattern in elevated CO; observed with tobacco represents a general phenomenon. However, it should be very clear that any future studies of acclimation that do not take this phenomenon into consideration in their experimental design and evaluation of results risk possible misinterpretation of their results.
In support of the temporal shift model as a more general phenomenon, other recent investigations of the interaction of leaf age with acclimation to elevated CO, in tomato (Van
Oosten et al., 1995), Rumex obtusifolius (Pearson et al., 1995) and wheat (Nie et al. 1995) are consistent with the temporal shift model, although these studies did not include data appropriate for critical evaluation of the model. Van Oosten and Besford (1995) observed significant down-regulation of photosynthesis under various high-CO, concentrations only after 60% leaf expansion, and therefore concluded that acclimation effects were seen only after a certain leaf age. However, unlike in the eariier paper on tomato (Besford et al. 1990), the "rate" of decline in CER during the fmal senescence phase of tomato leaf ontogeny was similar for all CO, concentrations, which is entirely consistent with a temporal shift model.
Nie et al. (1995) also concluded that acclimation in field-grown spring wheat was age-dependent and only occurred in older leaves. Again, their data are consistent with a temporal shift model, although they did not include leaf ages ^propriate to discriminate 36 between the authors' conclusions and the temporal shift model. Interpretation of the R. obtusifolius data in the context of the temporal shift model is complicated by the fact that
Pearson et al. (1995) measured CER under growth-CO, conditions, rather than under ambient
CO2, as in our study. Therefore, although they concluded that acclimation to elevated CO, resulted in an acceleration in the rate (rather than a shift in the timing) of the natural ontogenetic decline in photosynthesis, adjustment of their CER data to estimate rates under ambient CO, would make the pattern of decline roughly parallel to that of the ambient-COj- grown leaves, as would be predicted by the temporal shift model.
There is substantial evidence supporting the relationship between sink limitation and acclimation sensitivity (Stitt 1991). Therefore, if the temporal shift model is valid, the temporal shift in photosynthetic decline associated with acclimation may be dependent on a limitation in sink demand; if this were true, it would suggest that the normal progression of photosynthetic stages in leaf maturation is regulated by sink/source balance. In addition to support from the data presented here, interpretation of the Van Oosten and Besford (1995) data within the context of the temporal shift model also supports this suggested interaction.
Their data from four different growth COj concentrations indicate that the extent of the temporal shift of the photosynthetic decline appears to correlate with increasing source strength (increasing CO2 concentration) and therefore with decreasing sink/source balance.
Our results and those from some prior studies suggest that further investigation of the acclimation of plants to growth in elevated CO2 may even shed some light on the regulation of overall leaf development. Because the phase of photosynthesis decline normally is associated with progressive leaf senescence, the shift in timing of this photosynthetic phase may represent a temporal shift in the leaf senescence developmental pattern as well. In 37 combination with the apparent lack of any effect of elevated CO; on leaf expansion, this further suggests that control of these two leaf developmental phases or programs may be independent and uncoupled, with the timing of the senescence phase, but not the expansion phase, controlled at least to some extent by sink/source balance. Any firm conclusions about the interaction between sink/source balance and the shift in timing of developmental phases of leaf ontogeny will require further, directed investigations.
Acknowledgements
This work was supported in part by grants from the Carver Trust Foundation (to M.S. and S.R.) and from the Iowa State Biotechnology Council (to S.R.); and by an Iowa State
University Biotechnology Fellowship to A.M. This is journal paper no. J-17S2S of the Iowa
Agriculture and Home Economics Experiment Station, Ames, lA, and project no. 2987, and was supported by the Hatch Act and State of Iowa funds.
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photosynthetic apparatus in wheat, as indicated by changes in leaf proteins. Plant
Cell Environ 18:855-864 39
Pearson M, Brooks G (1995) The influence of elevated CO2 on growth and age-related
changes in leaf gas exchange. J Exp Bot 46: 1651-1659
Rodermel SR, Abbott MS, Bogorad L (1968) Nuclear-organelle interactions; nuclear
antisense gene inhibits ribulose bisphosphate carboxylase enzyme levels in
transformed tobacco plants. Cell 55:673-681
Sage RF (1994) Acclimation of photosynthesis to increasing atmospheric CO,: the gas
exchange perspective. Photosynthesis Research 39:351-368
Sicher RC, Kremer DF, Rodermel SR (1994) Photosynthetic acclimation to elevated CO2
occurs in transformed tobacco with decreased Ribulose-1,5-Bisphosphate
Carboxylase/Oxygenase content. Plant Physiol 104:409-415
Stitt M (1991) Rising CO2 levels and their potential significance for carbon flow in
photosynthetic cells. Plant, Cell and Environ 14:741-762
Stitt M, Schulze D (1994) Does Rubisco control the rate of photosynthesis and plant growth?
An exercise in molecular ecophysiology. Plant, Cell and Environ 17:465-487
VanOosten JJ, Besford RT (1995) Some relationships between the gas exchange,
biochemistry and molecular biology of photosynthesis during leaf development of
tomato plants after transfer to different carbon dioxide concentrations. Plant, Cell and
Environ 18: 1253-1266
Xu DQ, Gifford RM, Chow WS (1994) Photosynthetic acclimation in pea and soybean to
high atmospheric CO2 partial pressure. Plant Physiol lOi: 661-671 40
Ftgure Legends
Figiire 1. Leaf expansion and photosynthetic parameters during leaf ontogeny in ambient and
elevated CO,. O, Leaves grown under high-CO, conditions (950 ftL L'O- B. Leaves
grown under ambient-COj conditions (350 ftL L'O- Plants were moved into elevated
CO2 when leaf 10 reached 1 cm in length, but d-1 status was assigned to the leaf
when it reached 3 X 5 cm (approximately 5 d after exposure to elevated CO,). Each
point represents the average of measurements on leaf 10 for two to four individual
plants (means * SE); each plant was used for only one data point. The dashed
vertical lines indicate when peak CER values were attained for either high-CO, (d 4)
or ambient-CO, (d 12) treatment. A, Leaf width; B, net photosynthetic rate (CER); C,
Cs; and D, [Clj. All photosynthetic measurements were made under ambient- CO,
concentrations (350 /iL L'')-
Ftgure 2. Chlorophyll concentration and Rubisco concentration and activity during leaf
ontogeny in ambient (•)- and elevated (0)-C02 conditions. Experimental details are
as in the legend to Figure 1. A, Total chlorophyll concentration; B, initial Rubisco
activity; and C, total Rubisco activity.
Figure 3. Comparison between the down-regulation model and the temporal shift model of
high-COj acclimation. Dashed lines represent the down-regulation model, and bold
lines represent the temporal shift model. The inner arrows illustrate the difference in
perception as to how photosynthetic rate changes during acclimation. 41
25
M
IS I 1«
e
gi u
bi i.
U"
Relative Leaf Age (days)
Hguie 1. 42
500
300-
200-
100- a.
50-
M
O
100
75-
'cn g 50 *3
25-
Relative Leaf Age (Days)
Hgure 2. 43
Temporal Shift Model Normal Ontogeny
/
a: UJ o
Down-regulation Model
Time (days)
Figures. 44
CHAPTER 3. LEAF DEVELOPMENT AND ACCLIMATION
TO ELEVATED CO^
A chapter published in the Handbook of Plant and Crop Stress: Second Edition, Revised and
Expanded. New York: M Dekker. 1999
Adam Miller*, Martin Spalding*, and Steve Rodermel*
Introduction
Acclimation to Elevated CO,
Atmospheric CO; levels have changed dramatically since the Industrial Revolution,
increasing by almost 40% to a present-day concentration of approximately 350 ppm [1]. It is
thought that consumption of fossil fuels is the main contributor to this increase. The outlook
for the future is much the same, with the current level of CO, expected to almost double by
the beginning of the twenty-second century if existing conditions are not altered drastically.
How such changes in the atmosphere will affect the growth and development of plants, both
on an individual and global scale, is an important but unanswered question.
In many Q plants under normal environmental conditions, the photosynthetic rate is
limited by the rate of initial COj fixation into the Calvin Cycle. This rate-limiting step is
catalyzed by Rubisco, which carboxyiates ribulose-l, 5-bisphosphate (RuBP), generating two
molecules of 3-phosphoglycerate (3-PGA). Some 3-PGA molecules are exported into the
cytosol for use in sucrose biosynthesis, whereas others are responsible for RuBP regeneration
' Iowa State University, Ames, Iowa 50011 45
and starch synthesis in the plastid. Rubisco is an inefficient enzyme with a low affinity for
CO2, and it is substrate limited under present atmospheric concentrations [2].
One of the most important responses of plants to growth in elevated CO, is the
phenomenon of "acclimation" (reviewed in Refs.2-4). Because elevated CO, represents an
increase in substrate availability, increased rates of carboxylation in elevated CO2 should
result in higher net photosynthetic rates. In many experiments, this is observed in the short
term. However, the enhancement of photosynthetic performance is not maintained in plants
that "acclimate" to high COj, and in these plants photosynthetic rates fall below those
predicted on the basis of Rubisco kinetics. It is this loss of the predicted benefit of high CO2-
growth that is referred to as acclimation.
Acclimation has been observed in many agronomically important Q species, including tomato, wheat, pea, soybean, sugar beet, cotton, tobacco, and several tree species
[reviewed in Ref. 4]. However, Q plants differ in the severity of the acclimation response.
Also, C4 and erassulacean acid metabolism (CAM) plants do not acclimate, because they have biochemical carbon-concentrating mechanisms and are therefore constantly under an enriched CO, regime.
Molecular Mcchaniimi of AccliiiMitioB
Several mechanisms have been proposed to explain the downregulation of photosynthetic rates that occurs during acclimation (reviewed in Ref. 2). One mechanism is that enhanced starch accumulation in high- COj-grown plants results in large grains that disrupt chloroplast membrane structure and function. A second mechanism is that growth in enriched CO2 results in a reduction in stomatal conductance, which restricts the amount of 46
CO; entering the leaf; photosynthetic rates consequently fall [e.g., see Ref. S]. Yet a third
mechanism suggests that the decreases in photosynthesis during acclimation are a
consequence of enhanced rates of sucrose synthesis that accompany increased CO, uptake.
Enhanced sucrose production results in feedback regulation of sucrose phosphate synthase
(SPS) and a sequestering of Pi pools in the cytosol [2,6,7]. Without sufficient levels of Pi
returning to the chloroplast, RuBP regeneration, and hence photosynthesis, is restricted.
Finally, it has been suggested that a limitation in nitrogen may be a causal factor of
acclimation [8]. According to this mechanism, nitrogen assimilation is not able to keep pace
with enhanced photosynthetic rates under high CO,; that is, the photosynthetic mechanisms
are nitrogen limited.
Although all of these mechanisms may contribute to the acclimation response, recent
evidence has suggested that none can fully explain the long-term decreases in photosynthetic
rates that characterize this phenomenon. Rather, evidence favors the hypothesis that long-
term exposure to elevated COj results in a downregulation of photosynthetic gene expression.
One of the eariy pieces of evidence in support of this hypothesis was that declining
photosynthetic rates in high- COj-grown plants are accompanied by a loss of Rubisco protein
(e.g., see Refs. 1,2,7, and 9-15). In a few cases, it has been further demonstrated that the
loss of Rubisco protein is accompanied by coordinate decreases in Rubisco small subunit
(rbcS) and large subunit {rbcL) transcript levels in the nucleus-cytoplasm and chloroplast,
respectively [7,15].
Although alterations in rbcS and rbcL transcription may explain, at least in part, why
photosynthetic rates decrease during acclimation, there still remains the question of what factors control the changes in transcription of these genes. These factors are poorly A1 understood, but may include a variety of environmental factors such as nutrient status, water supply, mineral availability and temperature [16], which have been demonstrated to influence the sensitivity of the acclimation response. There also have been suggestions that acclimation can be influenced by leaf and plant developmental stage [14-16]. The latter is the focus of the rest of this chapter.
Elevated CO, and Tobacco Leaf Devdopmciit
Regiilation of Photosynthesis During Leaf Developnicnt
Under ambient CO2 conditions, leaf development can be separated into two distinct phases [17]. The first stage is associated with leaf growth and expansion. During this stage, leaf photosynthetic rates increase over time. There follows a transient peak of maximal photosynthetic rates correlated with the attainment of full expansion, and then rates begin to decline. This is the second stage of development, which is referred to as senescence. As the senescence phase progresses, leaves yellow as chlorophyll is broken down, and resources are reallocated to different parts of the plant, such as reproductive structures, in many species, the changes in photosynthesis that occur during these phases are largely due to changes in the levels of Rubisco protein and activity (e.g., see Refs. 18 and 19). The alterations in Rubisco protein, in tura, are due to coordinate changes in rhcS and rbcL mRNA amounts. This flnding emphasizes the notion that senescence involves a modulation of anabolic processes, as well as catabolic processes (cellular breakdown).
l>revious experiments in our laboratory have demonstrated that genetic manipulation of sink/source balance profoundly impacts plant growth and developmental processes [19, 48
20]. These experiments were performed with Rubisco antisense mutants of tobacco, in
which Rubisco levels are decreased up to 90% of those found in the wild type. Source
strength (carbohydrate production) is also impaired in these plants. We have also examined
growth and development under conditions where source strength is increased by growing
tobacco plants under elevated CO, conditions. These studies led to some novel observations
on the phenomenon of acclimation.
Photosynthetk Parameters
To investigate the effect of increased source strength on leaf development, we
examined tobacco plants under ambient CO3 levels (approximately 350 /ilA.) and enriched
CO2 concentrations (950 itUL) [21]. Leaf 10 (counting up from the base), which is a middle
canopy leaf, was chosen for analysis because of its large final size. The elevated CO,
regimen was initiated at the time of visible leaf 10 emergence. Measurements were taken at
varying time points throughout leaf development We first performed gas exchange analyses
and examined COj exchange rate (CER), stomatal conductance (Cs) and internal inorganic
carbon concentration (Ci). As illustrated in Figure 1, ambient COj-grown leaves exhibited
increasing CERs to day 12, a transient maximum, then a steady decline from day 14 onward;
rates fell below zero at day 40. Relative to their ambient-grown counterparts, the high-CO,-
grown leaves displayed a similar pattern of CER change over time, as well as a similar
photosynthetic maximum. However, these leaves reached their photosynthetic maximum and
initiated their photosynthetic decline at day 4, significantly earlier than in the ambient-grown leaves. The rate of photosynthetic decline following this maximum was comparable to that in the ambient-grown leaves, except that CER reached zero at day 25. These results suggest 49 that the magnitude and onset of maximal photosynthetic rates and their subsequent decline is similar in plants with enhanced source strength, but that in these plants, the onset of the decline is temporally shifted to an earlier initiation point.
To determine whether stomatal aperture was responsible for the changes in photosynthesis in high CO2 versus ambient-grown leaves, Cs and Ci were plotted versus relative leaf age [21]. The data indicated that there were no significant differences in Cs between the two treatments, but that the levels of Ci were generally higher in the elevated
COo-grown leaves throughout the developmental time course. Clearly, stomatal conductance did not cause the decline in photosynthesis observed in the high- C02-grown plants, since internal CO2 levels were not reduced. Considered together, these data demonstrate that CER was not limited by CO2 availability.
An examination of other photosynthetic parameters supported the conclusion that photosynthetic rates undergo a temporal shift to an earlier photosynthetic maximum in high-
CO^-grown leaves. In the first place, chlorophyll concentrations were similar between the two CO; concentrations early in development, but by day 6 chlorophyll amounts had already begun to decline in high- CO^-grown leaves; levels in ambient-grown leaves remained relatively constant until about day 16. The rates of chlorophyll loss during senescence were comparable between the two treatments. Second, the CER profiles were generally mirrored by similar changes in Rubisco activity and content in both sets of leaves. This suggests that photosynthetic rates may be determined primarily by Rubisco throughout leaf development 50
New Paradigm to Understand Accttmation
Temporal Shift Model
We propose a new model to explain the acclimation phenomenon. This model should be applicable to plants like tobacco whose major sinks are developing leaves. Much research over the years has supported the notion that there is a process that initiates the downregulation of photosynthesis via reductions in photosynthetic proteins after a certain length of exposure to elevated CO,. The end result is a loss of potential photosynthetic gain under favorable substrate conditions. This is especially evident when photosynthetic rates are measured under normal CO, conditions. Figure 2 illustrates this process.
To explain this downregulation, we have proposed a "temporal shift" model, which suggests that the lower photosynthetic rates observed after prolonged exposure to enriched
CO2 are due to an earlier onset of the natural ontogenic decline of photosynthesis associated with the senescence phase of development [21]. Figure 3 illustrates how our model differs from a photosynthetic downregulation model based solely on a change in the magnitude of photosynthetic rates that would occur during all leaf developmental phases.
Testing tiw Temporal Shift Modd in Rcdnccd Somve Strength Conditions
Elevated CO2 provides an easy method for increasing the source strength of a tobacco leaf. One way to test the validity of the temporal shift model is to decrease the source strength and examine the impact on photosynthetic rates during leaf development. One way that decreased source strength leaves has been achieved genetically in tobacco has been through antisense repression of Rubisco holoenzyme levels [22]. Rubisco is composed of 51 eight large subunit (LS) and eight small subunit (SS) proteins encoded by genes in the chloroplast {rbcL) and the nucleus irbcS), respectively. To generate the antisense mutants, a highly expressed member of the tobacco rbcS gene family was introduced into tobacco in the antisense orientation behind the highly expressed "constitutive" CaMV 35S promoter. The resulting transgenic plants had reduced rbcS mRNA and SS protein levels, and the reductions in SS were matched by corresponding reductions in LS and Rubisco holoenzyme amounts in the plastid, indicating that stoichiometric reductions occurred in the accumulation of these proteins in the mutant plants [22]. However, in contrast to rbcS mRNAs, rbcL mRNA levels were unperturbed in the mutants; it appears that LS accumulation is regulated primarily at the level ofrbcL mRNA translation initiation in these plants [23]. The reductions in Rubisco range from 10 to 90% of the wild type, and these reductions correlated with antisense copy number- the more rbcS antisense DNA present, the greater the repression of Rubisco. The reductions in Rubisco were accompanied by depressed photosynthetic rates, indicating that carbohydrate production (source strength) was also severely reduced in the mutant plants
[24].
To test the temporal shift model we have examined CERs and various other photosynthetic parameters during leaf development in the Rubisco antisense plants. We found that CERs are lower throughout development in antisense leaves than in leaves from either wild type or high-COs-grown plants (as expected). However, the onset of the decline in CERs associated with senescence occurred temporally later in leaf development in the mutants (data not shown). This is consistent with the temporal shift hypothesis. 52
Mcchanisin of the Temporal Shift
As illustrated by the above data, changes in leaf source strength appear to result in a temporal shift in the onset of the decline of photosynthetic rates associated with senescence.
Increased source strength caused a shift in photosynthesis to an earlier onset, while decreased source strength resulted in a delayed onset. This shift explains why, at a given leaf age, leaves from high-COj-grown plants have lower photosynthetic rates than their ambient- grown counterparts, such as observed in the tobacco acclimation studies of Sicher et al. [12].
The temporal shift model is consistent with the notion that photosynthetic output is determined by the sink status of the plant (sink regulation of photosynthesis) (reviewed in
Ref. 2). According to this theory, high photosynthetic rates are maintained in a source leaf as long as there is sufficient sink tissue (sink demand) to consume the carbohydrate that is produced by the source. However, once the demand for photosynthate falls, surplus carbohydrate begins to accumulate in the source. It is thought that this results in a long-term decline in photosynthetic rates due to feedback inhibition of photosynthetic gene expression.
Our temporal shift model is entirely consistent with the sink regulation hypothesis. Under increased source strength conditions (as long as the sink status remains unchanged), carbohydrate would accumulate more quickly, resulting in an earlier onset of the photosynthetic downregulation associated with sink limitation. Decreased source strength would have the opposite effect One possibility is that the changes in source strength are mediated by alterations in gene expression that occur by a sugar-signaling system similar to the catabolite-repression system of yeast (e.g., see Refs. 2.25,26).
There have been suggestions that the acclimation response is modulated by leaf developmental factors (e.g., see Refs. 14-16). The question arises whether the temporal shift 53 in photosynthetic rates is due to changes in photosyntbetic gene expression. Our data also raise the question whether the effects of source strength might be more broad and encompass other aspects of leaf developmental programming. We are currently investigating these questions.
References
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7. AN Webber, GY Nie, SP Long. Acclimation of photosynthetic proteins to rising
atmospheiic C02' Photosyn Res 39:413-425,1994. 8. H Nakano, A Makino, T Mae. The effect of elevated partial pressure of CO2 on the
relationship between photosynthetic capacity and N content in rice leaves. Plant
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10. S Yelle, RC Beeson Jr, MJ Trudel, A Gosselin. Acclimation of two tomato species to
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105: 441-448,1993.
12. RC Sicher, DF Kremer, SR Rodermel. Photosynthetic acclimation to elevated CO2
occurs in transformed tobacco with decreased ribulose-1,5-bisphosphate
carboxylase/oxygenase content. Plant Physiol 104:409-415,1994.
13. M Pearson, GL Brooks. The influence of elevated CO2 on growth and age-related
changes in leaf gas exchange. J Exp Bot 46: 1651-1659, 1995.
14. GY Nie, SP Long, RL Garcia, BA Kimball, RL Lamorte, PJ Pinter Jr, GW Wall, AN
Webber. Effects of free-air €02 enrichment on the development of the photosynthetic
apparatus in wheat, as indicated by changes in leaf proteins. Plant, Cell and Environ
18: 855-864,1995.
15. JJ VanOosten, RT Besford. Some relationships between the gas exchange, biochemistiy
and molecular biology of photosynthesis during leaf development of tomato plants after transfer to different carbon dioxide concentrations. Plant, Cell and Environ 18:
1253-1266,1995.
16. DQ Xu, RM Gifford, WS Chow. Photosynthetic accliniation in pea and soybean to high
atmospheric CO2 partial pressure. Plant Physiol 106:661-671,1994.
17. S Gepstein. Photosynthesis. In: LD Nood^n, AC Leopold, eds. Senescence and Aging in
Plants. San Diego: Academic Press, 1968, pp 85-109.
18. CZ Jiang, SR Rodermel, RM Shibles. Photosynthesis, Rubisco activity and amount, and
their regulation by transcription in senescing soybean leaves. Plant Physiol 101: 105-
112,1993.
19. CZ Jiang, SR Rodermel. Regulation of photosynthesis during leaf development in RbcS
antisense DNA mutants of tobacco. Plant Physiol 107: 215-224,1995.
20. CH Tsai, A Miller, M Spalding, SR Rodermel. Source strength regulates an early phase
transition of tobacco shoot morphogenesis. Plant Physiol 115:907-914,1997.
21. A Miller, CH Tsai, D Hemphill, M Endres, S Rodermel, M Spalding. Elevated CO2
effects during leaf ontogeny - a new perspective on acclimation. Plant Physiol 115:
1195-1200,1997.
22. SR Rodermel, MS Abbott, L Bogorad. Nuclear-organelle interactions: nuclear antisense
gene inhibits ribuiose bisphosphate carboxylase enzyme levels in transformed
tobacco plants. Cell 55:673-681,1968.
23. S Rodermel, J Haley, CZ Jiang, CH Tsai, L Bogorad. A mechanism for intergenomic
integration: abundance of ribuiose bisphosphate carboxylase small subunit protein
influences the translation of the large subunit mRNA. Proc Nat Acad Sci USA 93:
3881-3885,1996. 24. WP Quick, U Schurr, R Scheibe, ED Schuize, SR Rodermel, L Bogorad, M Sdtt.
Decreased ribuIose-13-bisphosphate carboxylase-oxygenase in transgenic tobacco
transformed with "antisense" rbcS. I. Impact on photosynthesis in ambient growth
conditions. Planta 183:542-554,1991.
25. A Krapp, B Hofmann, C Schafer, M Stitt. Regulation of the expression of rbcS and other
photosynthetic genes by carbohydrates: a mechanism for the "sink regulation" of
photosynthesis? Plant J 3:817-828,1993.
26. JC Jang, J Sheen. Sugar sensing in higher plants. Plant Cell 6: 1665-1679, 1994.
Figure Legends
Figure 1. CERs during development of tobacco leaves grown under ambient (circles) or
elevated CO, conditions (squares). Plants were moved into high COj when leaf 10
reached 1 cm in length; day "1" status was assigned when the leaf reached 3 X 5 cm
in length several days after transfer. Each point represents the average (± SE) of
multiple measurements on leaves from at least 4 different plants. (From Ref. 21.)
Figure 2. Downregulation of photosynthetic rates during a typical "accclimation"
experiment. Plants in ambient conditions (350 |iL/L CO,) are shifted to high CO;
(950 fiUL); controls are retained under ambient conditions. Measurements of
photosynthetic rates are conducted under ambient conditions after growth for varying
amounts of time.
Figare 3. The temporal shift model of acclimation. (From Ref. 21.) 57
Ambient HighCpj
1
gi U
Time(days)
Figure 1.
Growth xz Accllnfiated CER cz > if) o Transfer to high [cartwn dioxide] Time (days) Hgure2. 58 Temporal Shift Model Normal Ontogeny Down-regulation Model Time (days) Rgure 3. 59 CHAPTER 4. CARBOHYDRATE REGULATION OF LEAF DEVELOPMENT: PROLONGATION OF LEAF SENESCENCE IN RUBISCO ANTISENSE MUTANTS OF TOBACCO A paper published in Photosynthesis Research 63: 1-8 (2000) Adam Miller^-^, Cari Schlagnhaufer^, Martin Spalding'-'A Steven Rodermel'-^ Abstract Previous investigations have shown that increased source strength as a result of elevated CO, can alter the timing of the phases of change that occur in photosynthetic rates during dicot leaf ontogeny [Miller et al. (1997) Plant Physiol 115: 1195-1200]. To evaluate the converse situation of decreased source strength, we examined leaf development in rbcS antisense mutants of tobacco. These mutants have depressed Rubisco levels and decreased rates of carbohydrate production. We found that antisense leaves are longer-lived than wild type leaves and that this appeared to be due to a prolongation of the senescence phase of development, as monitored by photosynthetic rates, chlorophyll content, and the abundance and activity of Rubisco. Declines in these parameters during leaf ontogeny in both the wild type and mutant plants were generally accompanied by coordinate reductions in the levels of rbcS mRNA and rdcL mRNA, as well as reductions in chloroplast rRNA, chloroplast DNA ' Department of Botany, Iowa State University, Ames, Iowa 50011 ^ Interdepartmental Plant Physiology Major, Iowa State University, Ames, Iowa 50011 60 and total protein. We suggest that the prolongation of senescence in the antisense leaves is due to an impact of source strength on leaf developmental prognunming that occurs, at least in part, at the level of transcript abundance of nuclear and chloroplast genes for chloroplast rRNAs and proteins. We hypothesize that plants are capable of sensing a range of source strength conditions to initiate and modulate leaf developmental programming. Introdnctioa Leaf development is a highly ordered and complex process that is controlled and influenced by a variety of factors (reviewed in Van Lijsbettens and Clarice 1996; Brutnell and Langdale 1996). In general, dicot leaf ontogeny can be divided into three phases: an eariy phase of increasing photosynthetic rates when the leaf is actively expanding; a phase of maximal rates at full leaf expansion; and finally, a prolonged senescence phase of steady decline in photosynthetic rates (reviewed by Gepstein 1968). The senescence phase is mariced by a progressive yellowing of the leaf, loss of protein (most notably of Rubisco), and the translocation of resources to growing parts of the plant (Matile 1992; Lohman et al. 1994; Gan and Amasino 1997; Bleecker and Patterson 1997; Buchanan-Wollaston 1997). It has been suggested that carbohydrates play a central role in regulating leaf development (Jiang et al. 1993; Jiang and Rodermel 1995; Koch, 1996; Jang et al. 1997; Wingler et al. 1996). We have previously explored this possibility by investigating leaf ontogeny under conditions in which source strength (carbohydrate production) was increased by growing plants in elevated CO, conditions (Miller et al. 1997). These studies showed that the senescent decline in photosynthesis was initiated at an earlier time point in the high COj- grown leaves; these leaves began to senesce while they were still expanding. Other 61 characteristics associated with senescence, such as decreases in chlorophyll content, and in the content and activity of Rubisco, were also shifted to an earlier onset in the treated leaves. We suggested that the earlier onset of photosynthetic decline may explain, at least in pait, the down-regulation of photosynthesis (acclinution) seen in many Cj plants exposed to high CO, (Stitt 1991). We have also examined the impact of decreased source strength on plant development by taking advantage of Rubisco antisense mutants of tobacco. These mutants contain an antisense construct to the nuclear DNA-encoded (rbcS) small subunit (SS) of Rubisco. Expression of antisense rbcS RNAs in these plants results in the production of decreased sense rbcS mRNAs and reduced SS protein accumulation in the chloroplast (Rodermel et al. 1968). The levels of mRNA for the plastid DNA-encoded (rbcL) large subunit (LS) of the enzyme are unaltered in the mutant plants, and LS production is adjusted to that of the SS at the level of rbcL mRNA translation initiation (Rodermel et al. 1996; Rodermel 1999). Rubisco contents are reduced up to 90% in the various transformed lines (Rodermel et al. 1968), and the antisense plants are impaired in their ability to fix carbon and to produce carbohydrates suitable for export (Quick et al. 1991a, 1991b; Rodermel 1999). Jiang and Rodermel (1995) investigated various photosynthetic parameters in fully-expanded leaves from flowering antisense and wild type plants. They found that photosynthetic rates were lower in the mutants, but that in both types of plants, photosynthetic rates progressively declined as one moved down the canopy, i.e., from younger to older leaves. Also in the mutant and wild type, photosynthetic rates generally paralleled Rubisco contents and activities, as well as rbcS mRNA levels, suggesting that source strength exerts it effect during leaf development, at least in part, at the level of photosynthetic gene transcription. 62 Tsai et al. (1997) demonstrated that shoot development was profoundly altered in the Rubisco antisense plants. In particular, an eaily, slow-growth phase of shoot morphogenesis was markedly prolonged, and during this stage, more leaves than normal were also initiated. By contrast, the subsequent fast-growth phase of shoot development appeared normal, in terms of duration, rates of leaf initiation and leaf size. The antisense plants also had enhanced shoot/root ratios, and their leaves, regardless of nodal position, were longer-lived than normal. It was suggested that a threshold source strength is required to undergo the transition from slow-growth to the fast-growth phase, and that the alterations in whole plant and leaf development in the antisense plants are adaptations to maximize photosynthetic rates so that this threshold can be attained. The observations of Tsai et al. (1997) are consistent with the idea that the timing of developmental transitions, as well as the progression of leaf development, is significandy altered by changes in source strength. If source strength has a regulatory role during leaf development, we might expect a decreased source strength condition to have the opposite effect of increased source strength and to delay the initiation of the senescence decline in photosynthesis. We undertook experiments focusing on individual antisense rbcS tobacco leaves throughout their development to test this hypothesis. Matcrisli and Methods Pluit Material and Growth CowlkkMM For this study we used Rubisco antisense tobacco plants that contained ~20% of wild type enzyme amounts (Rodeimel et al. 1968). SRI tobacco plants served as controls. All 63 plants were grown and maintained in a conunon growth chamber, (IS-h photoperiods at approximately 300 ^mol photons m'^ s', 2S*'020°C and > 75% RH). Photosynthetic Mcararementi Photosynthetic rates (A), stomatal conductances and internal CO, concentrations (Cj) were measured using a closed chamber LiCor system, as described previously (Miller et al. 1997). Measurements were taken under growth chamber conditions on the middle third of the leaf surface. Three separate readings were taken at regular intervals and then averaged for a final value. Leaf discs (0.9 cm dia.) were punched out from the same area on which measurements for A were taken, then snap-frozen in liquid nitrogen and stored at -SOX until further analysis. Chlorophyll concentrations were determined using the method described by Amon (1949). For each individual plant, two of the frozen leaf discs were ground to a fine powder in liquid nitrogen and the pigments were extracted at 4"C using 80% acetone. Chlorophyll concentrations were then measured spectrophotometrically at A663 and A64S nm. Rubisco initial and total activity measurements were determined as in (Miller et al. 1997). Nucleic Acid Ana^nct Slot blot analyses were conducted with RNA and DNA samples. Procedures for extraction of nucleic acids and for slot blot hybridizations have been described previously (Jiang and Rodermel 199S). For the hybridization experiments, samples of denatured RNA and DNA (2^g) were loaded in random order onto a slot blot apparatus and transferred to GeneScreen Plus membranes. The membranes were hybridized overnight at 6S*C, then 64 washed and subjected to Phosphorlmage analysis (Molecular Dynamics). ImageQuant software was used to determine band intensities. Probes included tobacco rbcL and rbcS, the ChUunydomonas 16S rDNA, and the soybean 18S rDNA (all are described in Jiang and Rodermel 199S). The slot blots were normalized to J8S rRNA hybridizations. RcsaHi To determine the impact of reduced source strength on leaf development, we examined various photosynthetic parameters using leaves from wild type and rbcS antisense plants. The leaves were sampled throughout their development Our research group has previously found that an early, slow-growth phase of shoot development is specifically prolonged in the antisense mutants (Tsai et al. 1997). During this phase, more leaves than normal are initiated; however, the same number of leaves are initiated during a subsequent fast-growth phase (Tsai et al. 1997). Due to this early delay in developmental timing, we chose to compare leaves at node 13 of die antisense plants with wild type leaves at node 10. Both of these leaves emerge during the fast-growth phase and have similar characteristics (flnal size, canopy position, photosynthetic rate). Leaves 10 and 13 are thus considered to be 'developmentally similar'. We accorded 'Day T status to the leaves on the day when they reached at least 3 cm in width and 5 cm in length. This was a size sufficient for analysis. Figure 1A compares the rates of leaf expansion for leaf 10 in wild type tobacco plants versus leaf 13 in the antisense plants. The data show that both types of leaves have similar initial rates of expansion, and that they attain approximately the same size at full expansion. There were no measurements on wild type plants beyond day 40 because by this time leaf 10 65 had usually abscised. This did not begin to occur in leaf 13 of the antisense plants until approximately day 55. Hgure IB shows leaf dry weights as a function of the ontogeny of leaves from wild type and antisense plants. By the time they had attained full expansion, wild type leaves weighed two times more than developmentally similar antisense leaves. In both sets of plants, maximal weights were attained after the leaves bad stopped expanding. These data show that source strength influences leaf dry weight These differences may be due, in part, to differences in starch production, as previously observed in first fully-expanded leaves of the Rubisco antisense plants (Quick et al. 1991b). Figure IC shows that photosynthetic rates (A) increased to a maximum on day 12 in the wild type plants, then declined steadily until they fell below zero by day 40. Negative A values occurred when the respiration rate exceeded the photosynthetic rate, resulting in a net loss of carbon. Whereas maximal rates were somewhat lower in the antisense leaves, photosynthetic rates were relatively constant until about day 20, after which they declined steadily until day 30. Thereafter, they remained fairly constant and did not fall below zero, even at day 55. Figure ID shows that chlorophyll concentrations roughly changed in parallel with photosynthetic rates in both sets of plants. One exception to this generalization is that maxifnal chlorophyll levels were attained somewhat earlier in leaf development than maximal A values. Nevertheless, the striking feature of the data in Hgures IC and ID is that the senescence phase is markedly prolonged in the antisense plants. We next examined stomatal conductances and internal CO, (Q) concentrations (Figures 2A and 2B). No marked differences between the two source strength conditions could be discerned in stomatal conductances throughout most of development Q values 66 tended to be slightly higher in the mutants, consistent with the fact that these plants have greatly reduced levels of Rubisco and therefore cannot as readily consume the available COj. This greater availability of CO, may contribute to photosynthetic rates in the antisense leaves being closer to those found in the wild type than would normally be predicted. In general, however, these data show that throughout most of leaf development, photosynthetic rate differences between the wild type and antisense plants are not due to differences in stomatal aperture or CO, availability. Figure 3 shows initial and total Rubisco activity measurements. Rubisco initial activities represent the pool of activated enzyme in the leaf at the time of harvest. Total activities represent the total amount of activatable enzyme in the leaf and are proportional to Rubisco protein concentration. In both the wild type and antisense plants, Rubisco initial activities parallel total activities. However, as expected, Rubisco activities in the antisense leaves are significantly lower than in the wild type. In general, Rubisco was fully activated in the antisense plants, compared to about 50-75% activated in the wild type (data not shown). This is consistent with eariier observations on first fully-expanded leaves of the antisense plants growing under similar conditions (Quick et al. 1991a). With the exception of the large peak during the very eariy time points in the wild type leaves, the patterns of change in Rubisco activities and photosynthetic rates are similar. As a first approach to examine the factors that regulate Rubisco expression during leaf development, we isolated total cell RNAs from developing leaves and performed slot- blot hybridizations using rbcL and rbcS probes (Figures 4A and 4B respectively). The profiles of change in Rubisco subunit mRNAs during wild type leaf ontogeny are similar, and are generally consistent with the overall trends measured fm- Rubisco total activities - 67 i.e., an eariy peak followed by a decline. This suggests that coordinate changes in r^S and rbcL mRNA abundance play a central role in determining Rubisco content in the wild type plants. In the antisense leaves, the rbcS message is severely depressed, as expected (Rodermel et al. 1968), and the alterations in rbcL mRNA roughly match those in the wild type; both rbcS and rbcL mRNAs are maintained for a longer period in the mutants. These data suggest that the content of the holoenzyme in the antisense plants is regulated primarily at the level of rbcS transcript accumulation. This is in accord with flndings from antisense plants grown on tissue culture medium that LS protein accumulation is regulated post- transcriptionally at the level of rbcL mRNA translation initiation (Rodermel et al. 1968; Rodermel et al. 1996; Rodermel et al. 1999). One feature of senescence is that there is a loss of chloroplast polysomes and ribosomes, due either to active degradation and/or decreased synthesis (Matile 1992). Figure 4C illustrates that J6S (plastid) rRNA levels are lower in the antisense than wild type during early leaf development. However, they fall drastically in the wild type at the end of the sampling period, but remain steady from about day 25 onwards in the antisense leaves. For both the wild type and antisense, the patterns of change in 16S rRNA accumulation roughly parallel the changes in rbcL message levels. Southern hybridization analyses were performed to determine the profiles of change in plastid DNA to determine whether the alterations in r^L mRNA abundance are due, in part, to the availability of the polyploid chloroplast DNA template. Because it was difficult to accurately quantify and apply very small amounts of DNA from leaf discs to a slot blot filter, we normalized plastid DNA amounts K> nuclear DNA amounts by hybridizing the filter with an rbcL probe, then re-probing with a nuclear DNA sequence il8S iDNA). Relative 68 abundances were detennined by Phosphorlmager analysis and rbcVlSS rDNA ratios were calculated. The results are depicted in Figure 4D and show that chloroplast DNA levels are lower early in leaf development in the mutants, but that later they decrease in a similar manner in both sets of plants. Therefore, some of the loss of rbcL mRNA in both the antisense and wild type could be due to a decrease in rbcL DNA template availability. Difcusion According to the 'sink regulation of photosynthesis' hypothesis, photosynthesis is inhibited in source leaves by a build up of carbohydrates when sink demand is limiting (e.g., Stitt 1991; Sheen 1994; Van Oosten and Besford 1996). In agreement with this notion, a number of observations have shown that enhanced carbohydrate accumulation results in decreased photosynthesis and/or decreased photosynthetic gene expression (Sheen 1969; Krapp et al. 1991; Stitt 1991; Criqui et al. 1992; Krapp et al. 1993; Jang and Sheen 1994; Van Oosten and Besford 1995). These observations are also in accord with Koch's 'feast/famine' hypothesis (Koch, 1996) that insufficient carbohydrate levels induce the expression of genes whose products serve to increase carbohydrate supply (e.g., photosynthetic genes), while overabundance leads to a decrease in their expression. We and others have further suggested that feedback inhibition of photosynthetic gene expression by carbohydrates is an important factor that regulates the initiation of the decline in photosynthetic rates that characterizes the senescence phase of leaf development (Jiang et al. 1993; Hensel et al. 1993; Stitt and Sonnewald. 1995; Miller et al. 1997; Wingler et al. 1996). General support for diis idea comes from observations on trmsgenic plants with increased internal sugar levels (von Schaewen et al. 1990; Dickinson et al. 1991; Goldschmidt and 69 Huber 1992; Stitt and Sonnewald. 1995; Jones et al. 1996; Jang et al. 1997; Dai et al. 1999). These plants are stunted and have chlorotic or yellowed leaves with reduced rates of photosynthesis. Also consistent with this notion, enhanced rates of leaf senescence are observed when plants are grown in elevated light intensities (Veierskov 1967; Nood6n et al. 1996). Despite the attractiveness of the 'feedback inhibition' hypothesis to explain the progression and duration of the changes that occur in photosynthetic rates during leaf development, very few studies have directly investigated this hypothesis. We have previously examined various photosynthetic parameters during tobacco leaf development under increased source strength conditions (elevated COj) (Miller et al. 1997). Consistent with the feedback inhibition hypothesis, these experiments showed that the senescence phase was initiated and completed much earlier than normal in the high COj-grown leaves, but that the duration of this phase was unchanged in the treated plants. The effects of CO; are likely mediated, in part, by alterations in photosynthetic gene expression (unpublished observations). On the basis of these results, we suggested that the lower photosynthetic rates frequently observed during growth in elevated CO, ('acclimation') are the result, at least in tobacco, of a shift in timing of the normal photosynthetic stages of leaf ontogeny to an earlier onset of senescence (the 'temporal shift model') (Miller et al. 1997). Although there appear to be species-specific differences, these findings are in general accord with other studies that have investigated the impact of elevated CO2 on the progression of leaf development (e.g., Besford et al. 1990; Van Oosten et al. 1995; Nie et al. 1995; Pearson and Brooks 1995). The data in this report show that source strength regulates the duration and progression of leaf senescence. Our finding that both increased and decreased source 70 strength modulate leaf development suggests that leaf developmental programming is broadly responsive to a range of source strength conditions. One important element of this programming appears to involve the coordination of photosynthetic gene expression in the nucleus-cytoplasm and the chloroplast. Yet the responses we observed were not limited to the expression of photosynthetic genes, e.g., chloroplast rRNA and DNA abundance and total cell protein amounts (data not shown) were all affected by source strength. This suggests that source strength has a broad impact on leaf developmental programming, and is not merely limited to regulating photosynthetic gene expression. On the other hand, source strength does not appear to affect all of leaf development, as is evident by the similarity in leaf expansion rates both in this study and in our previous one (Miller et al. 1997). This may be a species-specific response, because elevated CO, results in increased expansion rates in some plants, while having little or no effect in others (e.g., Besford et al. 1990; Taylor et al. 1994; Sims et al. 1996). The mechanism by which source strength is sensed is likely to be complex. One current hypothesis is that hexokinase acts as a sugar sensor, initiating a signal transduction pathway that modulates the expression of various nuclear genes (reviewed in Jang and Sheen 1994; Koch 1996; Gan and Amasino 1997; Bleecker and Patterson 1997). Chloroplast genes for subunits of chloroplast multimeric protein complexes (e.g., LS and SS of Rubisco) also appear to be expressed coordinately in response to alterations in source strength during leaf development, and thus sugar sensing must involve regulatory communication between the nucleus and the plastid; these regulatory circuits are poorly deflned (reviewed in Rodermel 1999). One further complication is that factors other than carbohydrates, such as hormones and light, affect leaf development, and there is growing evidence that components of signal 71 transduction pathways for these factors interact and share elements in common (e.g., Zhou et al. 1996; Wingler et al. 1996). Our studies have shown that carbohydrates are able to regulate leaf developmental prognunming in a predictable manner, consistent with the idea of feedback inhibition of photosynthesis. We hypothesize that in some cases a threshold source strength is sensed and that this regulates a developmental switch— for instance, a phase transition in shoot morphogenesis (Tsai et al 1997) or the initiation of the senescence phase of leaf development (Miller et al. 1997). In this paper we show that source strength is able to modulate the duration of development responses once they have commenced. Acknowledgeiiieiits This work was supported in part by a grant from the Carver Trust Foundation (to S.R. and M.S.). This is journal paper no. J-186S9 of tiie Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa, and project no. 2967, and was supported by the Hatch Act and State of Iowa funds. Rcfcmcet Amon D1 (1949) Copper enzymes in chloroplasts. Polyphenol oxidase in Beta vulgaris. Plant Physiol 24:1-15 Besford RT, Ludwig LI and Withers AC (1990) The Greenhouse effect: acclimation of tomato plants growing in high CO2, photosynthesis and ribulose-1,5-bisphosphate carboxylase protein. J Exp Bot 41:925-931 Bleecker AB and Patterson SE (1997) Last exit: senescence, abscission, and meristem arrest in Arabidopsis. Plant Cell 9:1169-1179 72 Brutnell TP and Langdale JA (1996) Signals in Leaf Development. In: Advances in Botanical Research Vol. 28, pp 161-195. Academic Press Publishers, San Diego Buchanan-Wollaston V (1997) The molecular biology of leaf senescence. 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Plant Cell Environ 18:12S3-1266 Van Oosten J-J and Besford RT (1996) Acclimation of photosynthesis to elevated CO2 through feedback regulation of gene expression: Climate of opinion. Photosyn Res 48:3S3-36S Wingler A, von Scbaewen A, Leegood RC, Lea PJ and Quick WP (1996) Regulation of leaf senescence by cytokinin, sugars, and light. Plant Physiol 116:329-335 Zhou L, Jang J-C, Jones TL and Sheen J (1996) Glucose and ethylene signal transduction crosstalk revealed by an Arabidopsis glucose-insensitive mutant. Proc Natl Acad Sci 95:10294-10299 77 Figure Legends Figure 1. Changes in leaf expansion (A), diy weight (B), photosynthetic rate (C), and chlorophyll concentration (D) during leaf ontogeny in wild type and rbcS antisense mutants. Each point represents the average of measurements on up to four individual plants. Error bars are the standard error for each data point Because sampling for other analyses (described below) required destructive methods, younger leaves from the next node up the stem were harvested and used for leaf dry weight measurements (B). The wild type data (for A, B and C) are from Miller et al. (1997), and are included for comparative purposes. Figure 2. Changes in stomatal conductance (A) and internal (CO,] (B) during leaf ontogeny. Each point represents the average of measurements on up to four individual plants. Error bars are the standard error for each data point The wild type data are from Miller et al. (1997). Figure 3. Rubisco initial (A) and total activity (B) measurements during leaf ontogeny. The x-axis scale is different for Figures A and B. Each point represents the average of measurements on up to four individual plants. Error bars are the standard error for each data point The wild type data are from Miller et al. (1997). Figure 4i Changes in relative amounts of rbcS mRNA (A), rbcL mRNA (B), J6S rRNA(C) and rbcL ONA (O) during leaf ontogeny. Slot blot RNA hybridizations were standardized to 18S rRNA amounts. For the DNA hybridizations, approximately equal amounts of total cell DNA were applied to each slot, and the filters were hybridized successively with rbcL then 18S rDNA probes. Relative abundances were determined by Phosphorlmage analysis, and ratios of rbcL DUAJ18S rDNA were calculated. Each point represents the average of 78 measurements on up to four individual plants. Error bars are the standard error for each point Wild type 20 rbcS Antisense 15 10 2J •a OJ 7 J 400 200 Time (days) Figure 1. 80 0.4 —^ Wild type r^c5 Antisense OJ 0.1 — 4 500 400 300 200 100 Time (days) Figure 2. 81 Wild type 50 •••• rbcS Antisense 40 30 75 M \ SO- "3 25 0 10 20 40 50 Time (days) Figures. 82 100 —^ Wild type ..." r^5 Antisense a B €0 $ 40 20 V-i i.... 0 50 40 I 30 i 20 1 10 • -.4. 0 D 10 30 40 Time (days) Figure 4. 83 CHAPTER 5. CHANGE IN CARBON PARTITIONING DURING ARABIDOPSIS LEAF DEVELOPMENT A paper to be submitted to Photosynthesis Research for publication Adam Millet^'', Dan Stessman*^", Martin Spalding*-**, and Steven Rodermel*-" Abstract Previous investigations in our laboratory have shown that leaf developmental progranuning can be regulated by source strength. Increases in carbohydrate production led to an acceleration of the onset of the senescence phase in tobacco leaves, while decreased production caused a prolongation of the duration of this phase. Therefore, carbohydrates appear to have a key role in modulating leaf ontogeny. However, little is known about how carbon is partitioned on a leaf developmental scale. To address this question, we examined various photosynthetic parameters and carbon partitioning during the ontogeny of a single Arabidopsis thaliana (eco. Columbia) leaf (node #8). Chlorophyll contents, total soluble protein levels and photosynthetic rates declined progressively during development, even while leaves were still expanding. Carbon partitioning into sugars and starch reflected this pattern as well, however partitioning into ionic fractions (primarily amino acids) declined to a much lesser extent until the later stages of development The ratio of hexoses to sucrose increased up to full leaf expansion, then declined again. There is mounting evidence to suggest that carbohydrate flux through hexokinase may act as a sugar sensor to regulate the * Department of Botany, Iowa State University, Ames, Iowa 50011 ** Interdepartmental Plant Physiology Major, Iowa State University, Ames, Iowa 50011 84 expression of photosynthetic genes, possibly as a result of "futile cycling" of sucrose through vacuolar invertase. We found that the activities of hexokinase and invertase remained fairly constant until the very late stages of senescence, when they increased significantly. The data presented here provide a useful tool for future investigations into carbohydrate regulation of leaf development. Introdoction Previous investigations in our laboratory have shown that tobacco leaf developmental programming is regulated by source strength. Increased source strength resulting from elevated CO, environments caused an acceleration of the onset of leaf senescence (Miller et al., 1997), whereas decreased source strength in rbcS antisense mutants of tobacco resulted in a prolongation of senescence, and longer-lived photosynthetically active leaves (Miller et al., 2000). Analyses in other species have lent support to these flndings. For instance, developmental timing is altered when radish, barley, potato and Populus are grown under high COi conditions (Usuda and Shimogawara, 1996; Sicher, 1996; Ludewig et al.. 1996; Kauder et al., 2000; Wait et al., 1999). As another example, antisense Rubisco activase mutants of tobacco have greatly reduced carbon assimilation rates (Mate et al., 1993) and delays in the normal senescent decline of Rubisco (He et al., 1997). Effects on development of these plants could be partially compensated for by growth under enriched COj conditions (He et al., 1997). Impoitantly, studies of high CO2 acclimation in tomato have demonstrated that the response to source strength is sensitive to the degree of change in source strength (Van Oosten and Besford, 1995). This suggests that source strength effects on leaf development are an inherent mode of regulation, rather than a coarse response to a change in 85 carbohydrate production optimums. How these source strength effects are elicited however still remains unclear. Therefore, understanding how carbohydrates are distributed throughout the leaf may lead to a better understanding of how they act to regulate development. Newly fixed carbon from CO, has several fates in the leaf, most notably are the biosynthesis of different carbohydrates, amino acids and lipids. The degree to which carbon is distributed to each of these fractions is the partitioning. Determining the extent of carbon partitioning is an important factor when considering sugar signaling regulation of leaf development, since different carbohydrate fractions may result in different effects (Chiou and Bush, 1996; Geiger et al., 1998; Rook et al., 1998). Also, it has been suggested that carbohydrate flux rather than absolute pool sizes may be more important in regulating sugar responses in higher plants (Jang et al., 1997; Moore et al., 1997). Therefore, we examined partitioning over leaf ontogeny in the form of newly fixed carbon. In order to investigate carbon partitioning during leaf development, we first wanted to characterize the progression of leaf developmental photosynthetic parameters in Arabidopsis throughout ontogeny to provide the context Arabidopsis was chosen as a model due to the large knowledge base already present on this system, as well as the potential for future manipulation of sugar signaling components (Jang et al., 1997). We then measured carbon partitioning into several major fractions, as well as the changes in activity of hexokinase and invertase. This work will provide a body of background knowledge that will be useful in dissecting the mechanisms of sugar signaling regulation of leaf development. The results of these experiments are described here. 86 Materials and Methods Growth condJtioiis and Sample CoUecdon Wild type Arabidopsis thaliana (eco. Columbia) seeds were sown on soil saturated with Arabidopsis nutrient solution (Lehle Seeds "Methods for Arabidopsis thaliana" Communication) and vernalized in the dark for two days at 4^. The flats were transferred to growth rooms (constant illumination, ~100/iE at 20-2S''C) and sub-irrigated with water daily or when the topsoil became dry. Emergence of leaf 8 was noted and mariced with a small loop of colored thread for future identification. Sample leaves were removed from the plant at 20,24,27,35 and 40 days after germination. Sampling for invertase activity measurements were performed on days 16,18,20,22,24,27,30,33,36,39 and 42. The fresh weight of each leaf was measured before being snap-frozen and stored at -SO'C for future analysis. Hexokinaie Acthi^ Anays Hexokinase assays weie peiformed essentially as described by Huber (1969) and Renz et al. (1993). Leaf tissues were homogenized in SOO/iL of hexokinase extraction buffer (50mM MOPS, pH 7.5; 5mM MgCl,; ImM EDTA; 0.1% Triton X-1(X); 2mM Benzamidine; 2mM e-amino-o-caproic acid; 10% glycerol; 5mM DTT; 0.5mM PMSF; 2% PVPP) and centrifuged at 14,000 rpm for 10 minutes at 4*C. A 200;4L aliquot of the supernatant was desalted through a ImL Sephadex G-25 column equilibrated with elution buffer (50mM MOPS, pH 8.0; 5mM MgClj: 15roM KQ; 5mM DTT; 2mM Benzamidine; 2mM e-amino-n- caproic acid) by gravity. The resulting 400;iL protein-containing fraction was stored at 4*C until analysis. A 5|iL aliquot was removed and the protein concentration was determined 87 using the Bradford (1976) method. The remainder of the fraction was assayed for hexokinase activity in assay buffer (50mM MOPS, pH 8.0; 5mM MgCl,; 15mM KCl; SmM ATP; 0.5mM NADF; 2u G-6-P dehydrogenase) added to a total volume of ImL. Glucose was added to a concentration of ImM to start the reaction, which was assayed for NADPH production rate in a spectrophotometer at 340nm. Invcrtasc Assays Soluble (vacuolar) and insoluble (cell wall) invertase activities were determiued by procedures described by Huber (1989) for soluble invertase activities and Greiner et al. (1999) for insoluble invertase activities. Glucose amounts were measured spectrophotometrically using a glucose determination kit (Sigma Diagnostics). PigiiMBt Analysis Pigment extractions and concentration calculations were performed essentially as described by Lichtenthaler (1967). Leaf tissues were extracted with several changes of 95% ethanol in the dark at 4°C. Absorbance measurements were made at 664,649 and 470nm. Total SdaUe Protein Anatysb Total soluble protein concentrations were determined using the Bradford method (Bradford, 1976). The composition of the protein extraction buffer was as follows: ICXhnM Tris-HG (pH 7.5); 20mM KQ; 5mM EDTA; ImM PMSF; 5mM DTI and lOmM B- mercaptoethanol. Leaf tissues were homogenized in the buffer and allowed to stand on ice for 10 minutes. The samples were then centiifuged at 14,000 rpm for 10 minutes and the 88 supernatant was transferred to a clean tube for storage at -20°C. Several aliquots were measured at S9Snm and compared against a standard BSA curve according to the microassay procedure (Bio-Rad). RNA Isolatioii and Nmllieni Analytis Total RNA was isolated from leaf samples using the Purescript® RNA Isolation Kit (Centra Systems). Sfig of isolated RNA was mixed with an equal volume of 2X RNA Sample Buffer (50% deionized formamide; 16.5% formaldehyde; lOmM EDTA; 40mM sodium phosphate, pH 6.8; 0.2/ig/;4L Ethidium Bromide), heated at 65'C for 15 minutes, then quick-chilled on ice for 5 minutes. 2^L of RNA Blue juice (50% glycerol (v:v); 0.2% Bromophenol blue (w:v) in 5mM sodium phosphate, pH 6.8) was added to the tube and the samples were loaded onto a 3% Formaldehyde/1% Agarose gel for electrophoresis at 80 volts for 4-6 hours. The gel was then washed for Ave minutes in ddH^O five times. RNA was blotted onto a GeneScreen Plus membrane using the capillary transfer method. Hybridization procedures were performed as described previously in Jiang et al. (1993). Pbotosynthetk Rate and Carboa Partttkming Assays Photosynthetic rate and partitioning measurements were performed essentially as described in Sun et al. (1999). A closed system (see Figure 1) was used for radiolabeled COj feeding. *'^62 was released into the system by acidifying a NaH'^^O, solution (Amersham) with 5mL of 85% phosphoric acid. The solution was injected into a double-armed flask through a serum stopper to give a specific activity of O.lCi mol '. The was pumped through the system for at least 3 hours to equilibrate the system. A whole Arabidopsis plant 89 was placed into the cylindrical chamber and exposed for 10 minutes to the radiolabeled CO3 under constant light conditions (~125;4E). A 5% CUSO4 solution was used as a heat sink. After a 10-minute chase in unlabeled air, leaf eight was removed and weighed, and snap frozen in liquid nitrogen for future analysis. Before and after exposure to the plant, SmL gas samples were removed from the system with a syringe and injected into sealed vials containing ScintiGest tissue solubilizer to absorb the COj. These samples were allowed to stand overnight and then measured in a scintillation counter to determine the actual specific activity of the system for each plant. Labeled leaves were separated into soluble and insoluble fractions and measured for radioactive incorporation as described by Sun et al. (1999). The soluble fraction was separated further into neutral, cation and anion fractions by passing through ion exchange columns. Soluble sugar partitioning was determined using Thin Layer Chromatography (TLC) plates. A lO/iL aliquot was removed from the labeled soluble fraction and spotted onto a 20 X 20 cm TLC plate (K5 Silica Gel ISOA) (Whatman). The samples were run using 85% acetonitrile as a solvent for three separate ascensions. The plates were then dried and exposed to X-ray film for several weeks. The film was developed and spots corresponding to the migration of glucose, fructose and sucrose (identification based on comparison to several sugar standards run on a separate plate) were quantified using a densitometer. RmoHI To generate a developmental context within which to examine carbohydrate partitioning, we measured several general parameters that have been used as diagnostic indicators of the progression of leaf ontogeny. These are illustrated in figures 2 through 4. 90 Figures 2A and B show the expansion and change in fresh weight of leaf #8, respectively. Visible initiation was difficult to determine, so days after germination was used as a developmental time scale. The lack of significant variability in leaf expansion suggests that this method is consistent. Most sampling began on day 20, because these leaves were still in rapid leaf expansion, but were large enough to provide ample tissue for analyses. Full leaf expansion was attained between 24 and 27 days after germination, reaching a maximum size of around 18mm. As expected, Arabidopsis leaves gain fresh weight with continuing leaf expansion early on in development. After maximal expansion however, wild type leaves continue to gain in fresh weight to a maximum point around 35 days after germination. At this point the first visible signs of senescence (leaf yellowing at the tip) are clearly evident. After this peak fresh weights fall and by day 40 when leaves have clearly entered senescence, they have lost nearly a third of their maximal fresh weight. Presumably, diis is due a combination of reallocation of resources and a loss of water as the leaf begins to dry. Changes in photosynthetic rate, soluble protein and pigment contents are often used as markers for the progression of leaf development, specifically, progressive declines in these parameters are characteristic of senescence (Lohman et al., 1994). Figure 2C illustrates the change in photosynthetic rate during ontogeny. Photosynthetic rates show an initial maximal value, followed by a constant decline. Because measurements of photosynthetic rate are often made on a leaf area basis, we determined the leaf area to fresh weight ratio to confirm our results were valid. Although the ratio changes slightly with age, conversion of our data to a leaf area basis gave numbers reasonably similar to published values (data not shown). Figure 2D shows the change in total soluble protein over leaf development As with photosynthetic rates, soluble protein levels never showed a clear rise to a peak value. 91 Concentrations dropped steeply early in development, then declined steadily to the final data point Figure 3 illustrates the change in pigments over leaf ontogeny. Arabidopsis leaves show a large decline in chlorophyll content (Fig 3A) between days 35 and 40, which is consistent with visible observations of increased leaf yellowing over this period. However, chlorophyll contents show a steady decline from peak maximal values beginning from the first measurement on young non-fully expanded leaves similar to results seen in photosynthetic rate and soluble protein concentrations. Carotenoid contents also declined with leaf age (Fig 3B), and the degree of this decline from the maximum is similar to chlorophyll, as evident from the change in content expressed as a percent total basis (Hg 3C). The chlorophyll a/b redo (Fig 3D) remained fairly constant through most of development, until day 40 when the ratio decreased slightly. This suggests that although total chlorophyll contents decrease throughout development, preferential degradation of chlorophyll a versus chlorophyll b occurred in the later part of senescence. Changes in expression of cab and SAG 12 (SAG = senescence-associated gene) mRNA over development were also examined and these results are shown in Figure 4. The patterns of change observed are concordant with those reported for the Arabidopsis Landsberg ecotype (Lohman et al., 1994). Levels of cab expression decline with age, consistent with the results seen in the other photosynthetic parameters. Conversely, SAG12 levels increase dramatically duhng late senescence. These data suggest that changes in gene expression over development in Columbia and Landsberg ectoypes are similar. Newly fixed carbon in the forai of was used to determine the patterns of carbon partitioning over leaf ontogeny. Label was separated into insoluble (starch) neutral (sugars). 92 cationic and anionic fractions. The amount of total label incorporation into each fraction expressed on a nmol "^Oj/min/g fresh weight is illustrated in Hgure SA. The rate of incorporation into the neutral fraction showed a steady decline over development, paralleling the change in photosynthetic rate. Label incorporation into the ionic fractions declined only slightly during the first three measurements, but after day 27, the incorporation into this fraction decreased sharply. Figure 5B illustrates the change in carbon partitioning between the major soluble sugars over leaf ontogeny. A clear pattern is evident, with a gradual increasing proportion of label incorporated into the hexoses versus sucrose up to full leaf expansion, followed by a subsequent reverse of this trend. Late senescence resembles early development, with 90% or more of fixed carbon present in the soluble sugar fraction present as sucrose. The relative percents of each fraction are presented in Figure 5C. The general trends observed show an increase in partitioning into the cation fraction with leaf expansion, followed by a change to increased partitioning to the neutral (soluble sugar) fraction during the last two measurement periods. Therefore, the increase in partitioning into the cationic fraction through day 27 observed in Hgure SA is due to a relatively constant level of carbon being fixed in that fraction versus a greater decrease in carbon fixed in sugars and starch. This pattern may indicate the necessity for maintaining amino acid amounts (ionic fraction) early in development for anabolic purposes, and a switch in greater demand for energetic compounds during the extensive degradation processes that occur during senescence (Matile, 1992). Because hexokinase and vacuolar invertase have been implicated as having major roles in modulating carbohydrate signaling effects and may play a role in modulating leaf development, we investigated the change in activity of these two enzymes over development 93 The change in relative glucose phosphorylation activity over time is illustrated in Figure 6A. Although there are several enzymes that could potentially phosphorylate glucose: overall changes in activity are still indicative of changes in hexokinase because antisense hexoldnase mutants can decrease total glucose phosphorylation activities by over 65% (see Chapter 6, Figure 4). For most of the time points, hexokinase activity shows little change over development. However, at day 40 there is a dramatic increase in activity. The changes in soluble acid invertase activity depicted in Figure 6B show a very similar pattern. Discnssioii Surprisingly, the observed changes in the photosynthetic parameters measured did not match our expectations. Declines in chlorophyll content, soluble protein levels, and photosynthetic rate all suggest that leaf 8 of Arabidopsis initiated the senescence phase of development during the earliest stages of our measurements. It is quite possible that day 20 represents the transition from increasing to decreasing values, and the former was simply not observed due to the experimental time frame. This was not expected based on our tobacco studies (Miller et al., 1997; Miller et al., 20(X)). As with many dicot C3 leaves (reviewed in Gepstein, 1968), photosynthetic rates increased with leaf expansion in tobacco, and began their senescent decline after attaining full leaf expansion. The photosynthetic decline associated with senescence appears to occur before the attainment of full leaf expansion (Fig 2A). In the case of Arabidopsis, there may have been an increase to a peak occurring before our earliest data point. Nevertheless, we did not observe a clear initiatioa of senescent decline. However, our data are consistent with changes in photosynthesis observed in the fifth leaves of tomato (Van Oosten and Besford, 1996). Also, our results in Hgure 2D, 3 and 94 4 are in agreement with similar studies in fully expanded leaves of Arabidopsis Landsberg ecotype by Lohman et al. (1994) (corresponding to days 25-40 in the present study). Based on our current understanding of natural carbohydrate metabolism in the leaf, there are potentially two major sources of free hexoses that can serve as substrates for hexokinase signaling. These are breakdown of starch (Schleucher et al., 1998; Weber et al., 2000) and cleavage of sucrose by invertase or sucrose synthase (Foyer et al., 1968). In fact, species with high leaf activities of soluble acid invertase were more prone to exhibit an acclimation response to high CO, (Moore et al., 1996). This would suggest that hexokinase regulation of gene expression is strongly tied to "futile" sucrose cycling through invertase in the vacuole (Moore et al., 1999). Indeed, it has been observed that sucrose hydrolysis by different invertases may result in different pathway directions of the resulting hexoses in potato tubers as well (Hajirezaei et al., 2000). Therefore, the source of hexoses present in the leaf may be crucial to determining their impact on hexokinase sugar signaling. Further support can be found in examination of tomato SPS overexpressors grown under high CO^ conditions. Although higher glucose and fructose contents were seen in the transgenics versus the untransformed controls, Rubisco activities were of the same magnitude (Murchie et al., 1999). This suggests that increased levels of hexoses had no down-regulatory effect on photosynthesis in these plants. Similarly, hexose compaitmentation may also be a factor. It may be difficult to correlate soluble hexose pools with hexokinase sugar signaling under constant light conditions. It has been observed in Arabidopsis that hexose levels accumulate in the dark and may be mobilized during this period (Cheng et al., 1996). The relationship between hexokinase and hexoses may be more obvious under a diurnal cycle. 95 In Arabidopsis, glucose-pbosphorylating activities and acid invertase activities remain fairly constant throughout most of development until the later stages of senescence when they rise considerably (Figure 6). The changes in hexokinase activity with leaf ontogeny observed here are not universal however. In contrast, studies with tobacco have shown that hexokinase and fructokinase activities in both the particulate and soluble fractions declined with advancing leaf age (Sindel^ et al., 1997). The similarity between hexokinase activity and acid invertase seems logical, considering that sucrose hydrolysis in the vacuole is a potential major source of the hexose substrate. Why these activities rise so significanUy late in senescence is not yet understood and still requires further investigation. However, there is no clear correlation between changes in these enzymes and the changes observed in carbon partitioning during leaf development. Most surprisingly, partitioning into hexoses did not parallel with increases in invertase activity (Figure 5C). It is important to note that this does not preclude the possibility of in vivo factors altering the activity to correspond with sugar partitioning. At this time, it is not clear exactly why these activities should increase during late senescence. According to the partitioning data, there does not appear to be a large proportion of newly fixed carbon entering into the hexose fraction, but this does not preclude the existence of a large hexose pool present as potential hexokinase substrate. One possibility is that as a final step to resource reallocation, high levels of invertase and hexokinase activity are needed to remove any remaining sucrose pools present in the vacuole. The changes in development due to source strength alterations corresponded with similar changes in gene expression (Miller et al., 2000). Carbohydrate abundance has long been known to regulate the expression of certain genes, specifically those involved in metabolism (Graham, 1996; Koch, 1996). Only now however, are we beginning to elucidate 96 the mechanisms underiying carbohydrate gene regulation. While much is known about sugar signaling pathways in prokaryotes, yeast and mammals (Gancedo, 1992; Trumbly, 1992), plant systems are still poorly understood. There is recent evidence however, that strongly suggests that at least portions of these systems are conserved. Genes homologous to SNFl and SNF4, components of the yeast sugar-signaling pathway, have been identified in potato. Sorghum and Phaseolus (Man et al., 1997; Annen and Stockhaus, 1996; Abe et al., 1995). Their potential role in sugar signaling in higher plants is just now being explored (Purcell et al., 1996). The most complete studies have focused on the role of hexokinase as a potential sugar sensor in plants (Jang and Sheen, 1994; Jang et al., 1997; Smeekens and Rook, 1997). Flux through hexokinase was shown to regulate photosynthetic gene expression, and manipulating the degree of flux by altering enzyme levels resulted in corresponding increases or decreases in expression (Jang and Sheen, 1994; Jang et al., 1997). This makes hexokinase an attractive candidate for acting as a sugar sensor modulating the effects seen on development with altered source strength, especially considering the dosage effect witnessed under increased source strength conditions in tomato (Van Oosten and Besford, 1995). Although the experiments of Jang et al. (1997) suggested that hexokinase could act as a sugar sensor, the results were based on seedlings under excess carbohydrate conditions. Studies performed in tomato plants overexpressing Arabidopsis HXKl by Dai et al. (1999) provided new evidence that hexokinase does indeed play a role in regulating photosynthesis and growth throughout the life cycle of the plant, and under normal physiological conditions (endogenous sugar levels). Also, accelerated senescence was observed with a heterologous enzyme, suggesting conservation of sugar signaling pathways between plant species. 97 The model for hexoJdnase signaling is consistent with a role in feedback regulation of photosynthetic gene expression. However, it cannot be entirely ruled out that source strength effects on leaf development may be mediated via an alternative sugar-signaling pathway. The existence of sucrose specific (Chiou and Bush, 1998; Geiger et al., 1996; Rook et al., 1996) and other hexokinase independent (Roitsch et al., 1995; Martin et al., 1997) sugar signaling pathways have been demonstrated. Initial sugar sensing in these cases may involve membrane-linked molecules such as transporters rather than flux through hexokinase (Lalonde et al., 1999; Koch et al., 2000), maintaining the conservation seen between yeast and plant systems (Ozcan et al., 1996). In summary, Arabidopsis leaf development does not appear to follow the same pattern observed in tobacco. Photosynthetic parameters initiated their decline during leaf expansion, but the changes were still consistent with previous studies in Arabidopsis. Carbon partitioned into the total neutral (sugar) fraction paralleled this change. Partitioning into hexoses seemed to increase to approximately 25% of the neutral fraction at full leaf expansion, then dropped back down to less than 10%. This change did not correspond with changes in invertase or hexokinase activity in vitro. Future investigations into sugar signaling effects will hopefully benefit from this background information. Rcfcrenccs Abe H, Kamiya Y, Sakurai A (1995) A cDNA clone encoding yeast SNF4-like protein from Phaseolus vulgaris L. 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Plant J 11(1): 53-62 Mate CJ, Hudson GS, von Caemmerer S, Evans JR, and Andrews TJ (1993) Reduction of ribulose bisphosphate carboxylase activase levels in tobacco {Nicotiana tabacum) by antisense RNA reduces ribulose bisphosphate carboxylase carbamylation and impairs photosynthesis. Plant Physiol 102:1119-1128 Matile P (1992) Chloroplast senescence. In Crop photosynthesis: spatial and temporal determinants, NR Baker and H Thomas, eds. (Elsevier Science Publishers B.V.), pp. 413-441 Miller A, Tsai C-H, Hemphill D, Endres M, Rodermel S and Spalding M (1997) Elevated COj effects during leaf ontogeny: A new perspective on acclimation. Plant Physiol 115: 1195-1200 Miller A, Schlagnhaufer C, Spalding M, and Rodermel S (2000) Carbohydrate regulation of leaf development: prolongation of leaf senescence in Rubisco antisense mutants of tobacco. 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Plant Physiol Biochem 37(4): 251-260 Ozcan S, Dover J, and Johnston M (1996) Two glucose transporters in S. cerevisiae are glucose sensors that generate a signal for induction of gene expression. Proc Natl Acad Sci 93: 12428-12432 Purcell PC, Smith AM, and Halford NC (1996) Antisense expression of a sucrose non- fermenting-1-related protein kinase sequence in potato results in decreased expression of sucrose synthase in tubers and loss of sucrose-inducibility of sucrose synthase transcripts in leaves. Plant J 14(2): 19S-202 Renz A, Merlo L, and Stitt M (1993) Partial purification from potato tubers of three fructokinases and three hexokinases which show differing organ and developmental specificity. Planta 190:156-165 Roitsch T, Bittner M, and Godt DE (1995) Induction of apoplastic invertase of Chenopodium rubrum by D-glucose and a glucose analog and tissue-specific expression suggest a role in sink-source regulation. I^ant Physiol IM: 285-294 Rook F, Gerrits N, Kortstee A, van Kampen M, Borrias M, Weisbeek P, and Smeekens S (1996) Sucrose-specific signalling represses translation of the Arabidopsis ATB2 bZIP transcription factor gene. Plant J 15(2): 253-263 Schleucher J, Vanderveer PJ, and Sharkey TD (1996) Export of carbon from chloroplasts at night. Plant Physiol 118:1439-1445 103 Sicher RC (1998) Yellowing and photosynthetic decline of barley primary leaves in response to atmospheric CO^ enrichment. Physiol Plant 103:193-200 Sindel^ L, Sindelirovi M, and Burketovi L (1997) Hexokinases of tobacco leaves: influence of plant age on particulate and soluble isozyme composition. Biol Plant 40(3): 469-474 Smeekens S, Rook R (1997) Sugar sensing and sugar-mediated signal transduction in plants. Plant Physiol 115:7-13 Sun J, Okita TW, and Edwards GE (1999) Modification of carbon partitioning, photosynthetic capacity, and Oj sensitivity in Arabidopsis plants with low ADP- Glucose Pyrophosphorylase activity. Plant Physiol 119:267-276 Trumbly RJ (1992) Glucose repression in the yeast Saccharomyces cerevisiae. Mol Micro 6x 15-21 Usuda H, and Shimogawara K (1996) The effects of increased atmospheric carbon dioxide on growth, carbohydrates, and photosynthesis in radish, Raphanus sativus. Plant Cell Physiol 39(1): 1-7 Van Oosten JJ, and Besford RT (1995) Some relationships between the gas exchange, biochemistry and molecular biology of photosynthesis during leaf development of tomato plants after transfer to different carbon dioxide concentrations. Plant Cell Environ 18: 1253-1266 Van Oosten JJ, and Besford RT (1996) Acclimation of photosynthesis to elevated CO2 through feedback regulation of gene expression: Climate of opinion. Photosyn Res 48:353-365 104 Wait DA, Jones CG, Wynn J, and Woodward F1 (1999) The fraction of expanding to expanded leaves determines the biomass response of Populus to elevated CO;. Oecologia 121: 193-200 Weber A, Servaites JC, Geiger DR, Kofler H, Hille D, Groner F, Hebbeker U, and Fliigge U1 (2000) Identification, purification, and molecular cloning of a putative plastidic glucose translocator. Plant Cell 12:787-801 Figure Legends Figure 1. A schematic representation of the apparatus used to expose Arabidopsis thaliana leaves to for photosynthetic rate and carbon partitioning assays. Radiolabeled bicarbonate was injected into the serum cap-sealed double-armed flask containing phosphoric acid to liberate the which was then circulated by a pump. Figure 2. Changes in leaf expansion (A), fresh weight (B) photosynthetic rate (C) and total soluble protein concentration (D) during leaf ontogeny in wild type Arabidopsis thaliana (eco. Columbia) on leaf node 8. Leaf expansion measurements were initiated on day IS after germination and represent the average of 31 individual plants. In order to determine fresh weight, leaves were removed at the approximate petiole attachment position and weighed to the nearest mg before being snap-frozen for other analyses. Sample number ranged from 29 to 53 individual leaves measured per time point. Photosynthetic rates were determined on a fresh weight basis. Conversion of these data to a leaf area basis revealed values reasonably similar to published ones (data not shown). Each time point represents 5 to 6 samples. Total soluble protein concentrations were determined per mg leaf fresh weight, and sample 105 number ranged from 6 to 11 individual leaves measured per time point. All error bars represent the standard error for each data point. Figure 3. Qiange in Arabidopsis thaliana leaf 8 pigment contents over ontogeny. Pigment contents were determined per mg leaf fresh weight. Graph A represents the change in chlorophyll amount, (B) represents change in total carotenoids, (C) is the change in pigment content expressed on a percent total basis, and (D) represents the change in the chlorophyll a to chlorophyll b ratio. Error bars represent the standard error for each data point. Sample number ranged from 6 to 11 individual leaves measured per time point. Figure 4. Northern data demonstrating the change in mRNA expression of (A) cab and (B) SAG12 genes. Number labels represent the days after germination sampled. Sftg of total RNA was loaded in each lane and equal loading was conflrmed with ethidium bromide staining, as demonstrated in the picture provided (C). cDNAs were used to generate ^^P radiolabeled probes, and hybridizations were carried out under stringent conditions (65''C). Figure 5. Giange in partitioning of newly fixed into different fractions in Arabidopsis thaliana leaf 8 over ontogeny. Graph A is the total radiolabel incorporation into different fractions on a nmol '^Oj/min/g fresh weight basis. Black bars represent the insoluble fraction (starch), red represent the soluble neutral fraction, green the soluble cationic fraction, and blue the soluble anionic fraction. Error bars represent the standard error for each data point. Graph B is the change in carbon partitioning between sucrose, glucose and fructose. The black portion represents the sucrose percentage, the red portion is the glucose percentage and the green is the fructose 106 percentage. Each time point represents 5 to 6 samples. Standard errors ranged from 0.8 to 2.9% for sucrose, 0.6 to 1.4% for glucose and 0.5 to 1.8% for fructose. Graph C illustrates the relative percent partitioning between different soluble and insoluble fractions. The numbers present in the fraction are the actual percent values. Each time point represents 5 to 6 samples. Standard errors ranged from 2.2 to 3.8% in the starch fraction, 2.1 to 5.2% in the neutral fraction, 1.5 to 1.8% in the cation fraction, and 0.5 to 1.4% in the anion fraction. All colors remain consistent with A, except for the insoluble fraction, which is yellow in graph C. Figure 6. Change in relative glucose phosphorylating activity (A) and soluble acid invertase activity (B) of Arabidopsis lhaliana leaf 8 over ontogeny. Values in A are relative rates measured on a per mg protein basis. Invertase values (B) represent / glucose formed per mg protein per hour. Error bars represent the standard error for each data point. Sample number ranged from 4 to 10 individual leaves measured per time point. 107 /—— > I 11 / \ 14C0 Hgure 1. Total Sol Botein 0«AngFW) Fresh Weight (mg) Leaf Width (mm) 2 S ^ s 8; s _i I i_ S w 109 2 1.5 1 OJ 0 0.4 0.9 0.2 0.1 0 120 100 80 40 Chlorophyll 20 Carotenoids 0 3 D 2 1 0 I I I I I 15 20 25 30 35 40 45 Time (days after germinatioii) 110 20 24 27 35 40 CAB B 20 24 27 35 40 SAG12 20 24 2735 40 ft Figure 4. Ill Rj bmluHe Ncuml • Ciliaa S Amoa • Sucrow • Glucow • Finictaw M 24 27 35 40 Time (days after germination) d20 d24 d27 d3S d40 • Insoluble (Starch) • Neutral (Sugars) • Cations • Anions Figures. 21 Average Soluble Add Average Relative Invertase Activity Hexokinase Activity (|imol/mg protein/hr) (rate/mg protein/hr) w * Ul I I .;.w-X-;-XvXvX'X'l H SS 3 IIIi III ITTmTTT K> & 55 •n OQ 8»" "• S v.'.'.y .•.•.•.•.•.•.•.•••.•J • I III tt • I II .•.•••••.•.•••••.•.•.•.•.••••nv.'.'.'.N'.'.'.'.'J o ^•^•^«^»eeee^^5%^»^e^e%ee^Fei^eee1 9 $ '.%'.%%%%%%*>%V,\»,%%'||•••••••••••••• • • • • I • I'l'lJ• • • DO ft '*'*'Vi*i*fYiyM*iYi*i'i*i*iyiv*iV'*'*'''''*'*'*-*'*'*'*'*'''*'*'*' 113 CHAPTER 6. GENERATION OF TRANSGENIC ARABIDOPSIS WITH ALTERED LEVELS OF HEXOKINASE (HXKl) ACTIVITY Introdoctioii Transgenic plants with altered levels of hexokinase activity have already been generated in a variety of plant species, including tomato, potato and Arabidopsis (Dai et al., 1999; Veramendi et al., 1999; Jang et al., 1997). In order to fully investigate the role of hexokinase sugar signaling on leaf development and senescence, we proceeded to generate transgenic plants with both increased and decreased levels of HXKl activity under the transcriptional control of a cabS leaf specific promoter. A description of the generation of these mutants is provided here. Materiali aad Mcdiods Growth Conditkuii and Sample CoUcetioo Wild type and transformed Arabidopsis thaliana (eco. Columbia) seeds were sown on soil saturated with Arabidopsis nutrient solution (Lehle Seeds Methods for Arabidopsis Thaliana) and vernalized in the dark for two days at 4°C. These flats were transferred to growth rooms and maintained under constant light conditions (~100^E). Temperature was regulated between 20-2S°C. Germination occurred two days later. Flats were sub-irrigated with water daily or when topsoil became diy. Emergence of leaf 8 was noted and marked 114 with a small loop of colored thread for future identification. The fresh weight of each leaf was measured before being placed in a labeled microcentrifuge tube and snap-frozen in liquid nitrogen. These samples were then stored at -80'C for future analysis. In order to obtain substantial amounts of root tissue to test the tissue specificity of the cab3 promoter, wild type and HXKl overexpressing plants were grown hydroponically based on the description provided in Gilbeaut et al. (1997). 50mL conical centrifuge tubes were used, and a small opening was made in the cap with a cork borer. A Rockwool (GrodanHP, Agro Dynamics, East Brunswick, NJ) plug was inserted into the cap opening to act as the rooting media. The tube was filled with nutrient solution and seeds were sown on the top of the plug. Tissue was harvested a few days after bolting. Vcclor Constroction and Plant Transformation Dr. Jyan-Chun Jang kindly supplied the Arabidopsis AtHXKl clone (present in the pBIuescript vector). The cab3 promoter was amplified from Arabidopsis genomic DNA using standard PGR methods. Primers were designed based on the promoter sequence information from Mitra et al. (1969), and to include restriction sites at either end to facilitate sub-cloning into a modified pBll 1IL transformation vector containing the NFTII gene conferring kanamycin resistance (Primer sequences: 5' CCA AGC TTC CAA TGA TGT TGA ACA TACC 3' and 5' GGT CTA GAT TGT GGA GGC GGC GAT TGA AAC 3'). The PGR product was digested with the appropriate restriction endonucleases and was electrophorescd in a 0.8% agarose gel. The band was excised from the gel and the DNA purified using the Qiagen DNA Gel Extraction kit This fragment was ligated into the digested pBI 11IL vector and the new vector was renamed pBIC. Generation of the 115 overexpression construct is as follows. The 2kB Not I fragment of AtHXKl was digested from the original vector and purified as described previously. A fill-in reaction was carried out to generate blunt ends on the Not I fragment This fragment was then ligated into the Sma I cloning site of pBluescript This manipulation was performed in order to provide the proper restriction sites for ligation into the pBlC cloning site. Generation of the antisense construct began with a digestion of the HXKl clone with Sstl. The resulting fragment was ligated into the Sstl site in the pBIC vector and tested for antisense orientation with a EcoRI digestion. The new vectors (see Hgure 1) were amplified in E. Coli and were introduced into Agrobacterium tumifaciens via electroporation. Transformation of Arabidopsis was carried out essentially as described in Bent and dough (1998), and Qough and Bent (1996). Sterile T1 seed was plated on MS media containing 50/4g/mL kanamycin for selection of putative transformants. These seedlings were transferred to soil, seed collected and progeny tested for insert number, altered hexokinase mRNA levels and glucose phosphorylating activity. Antisense transgenics were placed under a secondary sugar sensitivity screen. Sugar Scnsitivi^ Scrcen for Antiscnw Hexokinase Mntants Conditions for the sugar sensitivity screen were adapted from Jang et al (1997). T2 seed from kanamycin insensitive plants transformed with the antisense HXK construct were sterilized and spread on half-strength MS plates containing 6% glucose. The plates were wrapped with Parafilm and placed at 4*C for two days. After vernalization, the plates were unwrapped, dried and then placed under constant light conditions. Seedlings that cleariy demonstrated glucose insensitivity (green, expanded cotyledons) were transferred daily to plates with progressively decreasing concentrations of glucose (4,2 and 1%) to prevent 116 osmotic shock. These seedlings were then transplanted to soil and under normal growth conditions to collect seed. Progeny were tested for HXKl message and activity. Figuie 2 outlines the selection strategy for both transgenics. DNA and Soothcrn Analyiit Genomic DNA was isolated from fully-expanded leaf tissue as described previously. Roughly 5fig of DNA was digested with the Bgl II restriction endonuclease to produce a single band for the endogenous gene and any inserted copies. Agarose gels were electrophoresed, blotted and hybridized essentially as described previously (Jiang et al., 1994). The Not I fragment of the AtHXKl clone was used as a probe. RNA Isobtion and Northern Ana^ysii Total RNA was isolated from leaf samples using the Purescript® RNA Isolation Kit (Centra Systems). Sftg of isolated RNA was mixed with an equal volume of 2X RNA Sample Buffer (50% deionized formamide; 16.5% formaldehyde; lOmM EDTA; 40niM sodium phosphate, pH 6.8; 0.2;4g/;iL Ethidium Bromide), heated to 65°C for 15 minutes to denature, then quick-chilled on ice for 5 minutes. 2/iL of RNA Blue juice (50% glycerol (v:v); 0.2% Bromophenol blue (w:v) in 5mM sodium phosphate, pH 6.8) was added and the samples were loaded onto a 3% Fonnaldehyde/1% Agarose gel for electrophoresis at 80 volts for 4-6 hours. The gel was then washed for five minutes in ddHjO five times. RNA was blotted onto a GeneScrcen Plus membrane using the capillary transfer method. Hybridization procedures were performed as described in Jiang et al. (1999). The SsU fragment of the HXKl cDNA clone was used as a probe. 117 Hexokinase Activity Assays Hexokinase assays were performed essentially as described in Huber (1969) and Renz et al. (1993). Protein samples were prepared from ground leaf tissue in 500/ 2mM Benzamidine; 2mM E-amino-n-caproic acid; 10% glycerol; 5niM DTT; 0.5mM PMSF; 2% PVPP) and centrifuged at 14,(X)0rpm for 10 minutes at 4''C. A 2(X)/4L aliquot of the supernatant was desalted through a ImL Sephadex G-25 column equilibrated with elution bufTer (50mM MOPS, pH 8.0; 5mM MgCU; 15mM KQ; 5mM DTT; 2mM Benzamidine; 2mM E-amino-n-caproic acid) by gravity. The resulting ACOfiL protein-containing fraction was stored at 4°C until analysis. A S/iL aliquot was removed and the protein concentration determined using the Bradford (1976) method. The remainder of the fraction was assayed for hexokinase activity in assay buffer (50mM MOPS, pH 8.0; SmM MgQ,; 15mM KCl; SmM ATP; O.SmM NADP; 2u G-6-P dehydrogenase) to a total volume of ImL. Glucose was added to a concentration of ImM to start the reaction, which was assayed for NADPH production rate in a spectrophotometer at 340nm. Results And Discasskm Figure 3 shows the Southern results on the progeny of both the overexpression and antisense transformants. Wild type samples are labeled "WT", while all other samples are transformant progeny. A sample from the overexpression progeny (labeled OE) was included in the antisense Southern for comparative purposes. As is indicated by the bloc, all lanes had the endogenous HXKl band present A second larger fragment in a number of the lanes indicates a single T-DNA insert The lack of a second band in some of the 118 transfoimants suggests the parents were hemizygous and the insert has segregated according to Mendelian genetics. Progeny were then tested for glucose phosphorylating activity. Samples indicated with an asterix also showed altered glucose phosphorylating activity (see Figure 2), indicating that the single insert is responsible for changes in activity. Figure 4 shows the relative glucose phosphorylating activity data from the overexpression and the antisense progeny. As indicated by the graphs, there are clear differences in relative activity between wild type and the transformants. Overexpression mutants showed up to 10-fold more activity in their leaves compared to wild type, while antisense leaves demonstrated as much as a 67% reduction in activity. In both cases, alterations in activity corresponded with the presence of a second band on the Southern analyses (see Hgure 1), suggesting that these differences are due to the presence of the introduced construct. Figure 5 shows the relative HXKl mRNA abundance, as determined through Northern analysis. A picture of the gel is provided to confirm equal loading. From this data it is clear that changes in glucose phosphorylating activity seen in the transgenic progeny are due to alterations in the abundance of HXKl mRNA specifically. As with activity, changes in message abundance corresponded with the presence of an insert Overexpression leaves (in blue) contain high message levels compared to wild type (labeled WT), whereas antisense leaves (in red) have greatly reduced levels. The smaller band present in the antisense sample lanes is due to the presence of antisense mRNA picked up by the probe. Progeny with the most striking alterations in hexokinase activity were chosen for the purpose of isolating homozygous lines. The reasoning behind this was that a homozygous line would have double the copy number of a hemizygous one, and therefore would likely 119 have more pronounced changes in activity. Seed from these plants were collected and seedlings were initially screened for the presence of the NFTII gene conferring kanamycin resistance via PCR methods. These results were then confirmed with Southern analysis. All the progeny tested in the overexpression mutant contained the insert, strongly suggesting the line was homozygous (data not shown). Antisense lines are in the process of being tested in a similar manner. The transgenics generated here will be used for further analyses testing the role of hexokinase as a sugar sensor in higher plants. According to our model of source strength regulation of leaf development, we would expect changes in signal generation potential to act in a similar manner to that of altering carbohydrate production. Increased hexokinase activity would represent increased signal generation (as long as sugar production isn't limiting), and we would expect a response similar to the one observed under increased source strength; acceleration of onset of senescence (Miller et al., 1997). Conversely, we would expect decreased activity to result in developmental effects similar to decreased source strength; prolongation of senescence duration (Miller et al., 2000). Although examination of changes in carbon partitioning did not reveal any obvious correlation with changes in ontogeny, this data will be useful in future analyses regarding source strength regulation of leaf development. We will compare the change in photosynthesis and senescence related parameters over leaf ontogeny that have been documented in wild type (Chapter 5), with changes observed in these mutants. These results should provide valuable insight into the mechanism of source strength developmental regulation, as well as the role of hexokinase as a sugar sensor under noimaJ physiological conditions. 120 References Bent AF and Qough SJ (1996) Agrobacterium germ-line transformation: Transformation of Arabidopsis without tissue culture. In Plant Molecular Biology Manual (Gelvin, S.B. ed.)- Netherlands: Kluwer Academic Publishers, B7: 1-14 Clough SJ and Bent AF (1996) Floral dip: A simplified method iot Agrobacterium-mtAxzvtA transformation of Arabidopsis thaliana. Plant J 16(6): 735-743 Dai N, SchafTer A, Petreikov M, Shahak Y, Ciller Y, Ratner K, Levine A and Granot D (1999) Oerexpression of arabidopsis hexokinase in tomato plants inhibits growth, reduces photosynthesis, and induces rapid senescence. Plant Cell 11: 1253-1266 Gilbeaut DM, Hulett J, Cramer GR, and Seeman JR (1997) Maximal biomass of Arabidopsis thaliarui using a simple, low-maintenance hydroponic method and favorable environmental conditions. Plant Physiol 115:317-319 Huber SC (1969) Biochemical mechanism for regulation of sucrose accumulation in leaves during photosynthesis. Plant Physiol. 91:656-662 Jang J-C, Ledn P, Zhou L and Sheen J (IS^ Hexokinase as a sugar sensor in higher plants. Plant Cell 9:5-19 Jiang CZ, Rodermel SR and Shibles RM (1993) Photosynthesis, Rubisco activity and amount, and their regulation by transcription in senescing soybean leaves. Plant Physiol. 101:105-112 Miller A, Tsai C-H, Hemphill D, Endres M, Rodermel S and Spalding M (1997) Elevated CO2 effects during leaf ontogeny: A new perspective on acclimation. Plant Physiol IIS: 1195-1200 Miller A, Schlagnhaufer C, Spalding M, and Rodermel S (2000) Carbohydrate regulation of leaf development: prolongation of leaf senescence in Rubisco antisense mutants of tobacco. Photosyn Res 1-8 Mitra A, Choi HK, and An G (1969) Structural and functional analyses of Arabidopsis thaliana chlorophyll a/b-binding protein (cab) promoters. Plant Mol Biol 12:169- 179 Renz A, Merio L and Stitt M (1993) Partial purification from potato tubers of three fructokinases and three hexokinases which show differing organ and developmental specificity. Planta 190:156-165 Veramendi J, Roessner U, Renz A, Willmitzer L, and Trethewey RN (1999) Antisense repression of hexokinase 1 leads to an overaccumulation of starch in leaves of transgenic potato plants but not to significant changes in tuber carbohydrate metabolism. Plant Physiol 121:123-133 122 HinD III. Ttrminator LtftBordtr Promottr Rifht pBIC AtHMCI 13.00 Kb HinD III. NOS T«rmlMtor I ieo Rl Uft Border Pron»t#r Right ^IC AntiHXKI 15i»Kb Hgure 1. A graphic representation of the vectors used for Arabidopsis transformation. The top vector was used to generate HXKl overexpressor transgenics, and the bottom vector was used to create antisense HXKl transgenics. The original vector was a modified pBl 121 with the GUS gene digested out (renamed pBIl 1IL). The 3SS promoter was digested out and replaced with the cab3 promoter PCR product using the restriction digest sites indicat^ in the figure, and the resulting plasmid was renamed pBIC. 123 Wild Type Plant (TO generation) J Floral Dip Transformation Collect Seed (T1 generation) Overexpression Construct Antisense Construct I Plate on Kanamycin I Transfer and PGR Screen T1 Survivors Plate on 6% Glucose 1/2 MS plates Collect seed from PCR-t- plants (T2 generation) Transfer and PGR Screen T1 Survivors I Choose several T2 seed and Collect seed (T2) from a survivor plant flats and test progeny: Southern, Northern, Activity I SEE FIGURES 3-5 Test several plants on each flat for high hexokinase activity I Collect seed (T3) from an overexpressor and test progeny: Southern, Northern, Activity SEE FIGURES 3-5 Hgure 2. A flowchaft outlining the selection procedures used for isolating cverexpiession and antisense transgenics. 124 IB8-A8 (Overexpressor) B4 B6 C2 C3 C4 D9WT * * « « —-Antisense IC7 from plates--— A1 A2 A3 A4 A5A6 A7 A8A9 OEWT t * * * * * « Rgure 3. DNA Southeni results on T3 transgenic progeny. The top film is from an overexpressing line and the bottom film is from an antisense line (including one overexpressor as a control). Each Southern represents examination of progeny of a single transformation event. The letter-number designations correspond with the designations on the activity figures (Figure 4). The presence of a single band indicates the endogenous gene (as in the wild type lane), while a second band indicates the presence of the inserted gene. The data show the parents were single insert, hemizygous plants. Lanes mariced with an asterix indicate progeny that also displayed altered enzyme activities (see Figure 4), demonstrating that the insert was responsible for altered activities. 125 • Wild Type Q Overexpressor T3 progeny Puent: line IB8-A8 1 I * f i| Hi I. WTAlAlAaA4Af AtfASAf BtB2KIB4BfBiB7nWClClC3C4C7CtC»DlDai>3MD7Df Plant Genotype E3 Wild Type B [31 Antisense T3 progeny I^nt: line IC7 I: \ \ ^ s' > ' / • N• ^ \ ^ • / / / \ > s • ' • f / ' \• N 'S • \ • » \V ^ s WT A1 A2 A3 A4 AS Ai A7 A8 Plant Genotype Figure 4. Graphs of relative glucose phosphorylating activity on T3 overexpressor (A) and antisense (B) progeny. Activity was determined as a relative rate of NADP reduction per mg protein. Each bar represents an individual plant and all plants are progeny from a single transformation event Activity patterns are indicative of single insert, hemizygous parents. 126 WT A9 B2 B3 B6 B8 A3 A5 A8 D1D6 D7 D9 A9 • Hguie 5. Northern blot results of wild type, overexpressor, and antisense transgenic progeny HXKl mRNA. Overexpressor progeny are designated in blue, and antisense progeny are designated in red. The lower band in the antisense lanes is hybridization of the probe to the antisense mRNA. A picture of the gel is included below for confirmation of equal lane loading. 127 CHAPTER 7. GENERAL CONCLUSIONS AND FUTURE RESEARCH DIRECTIONS Conclusions and Fbtnre Rcscareh Conclusions My dissertation proposal states that the timing of certain leaf developmental processes is regulated by the production of sugars, and that this control is mediated through the activity of the hexokinase enzyme. To begin testing my model of carbohydrate regulation of leaf development, 1 conducted experiments designed to alter source strength conditions and observe the changes on development. In leaves, source strength refers to the ability of a leaf to produce and export photoassimilates (Ham et al., 1993). Therefore a change in source strength would alter the carbohydrate status of the leaf. We used two different model systems in tobacco plants to create these situations. First we investigated increased leaf source strength. In order to increase source strength, we grew our plants under enriched CO2 concentrations (Miller et al., 1997). Elevated carbon dioxide levels allow for a greater carboxylation rate by Rubisco, and in the short-term results in a stimulation of photosynthetic rate (Stitt, 1991). This increase in photosynthetic rate has also been demonstrated to lead to an increase in carbohydrate production, including free hexoses (Nie et al., 1995; Van Oosten and Besford, 1995). If the sink limitation hypothesis is accurate, sink demand in tobacco would eventually not be able to support the increase in source output, resulting in a feedback on supply and an accumulation of carbohydrates in the source leaf. 128 Photosynthesis would then be down regulated. In fact, repression of photosynthetic gene expression under high COj has been shown to be similar to repression due to glucose feeding (Van Oosten et al., 1994). Several developmental parameters were measured over the life of a single tobacco leaf. A description of the methods employed can be found in Miller et al (1997). The results obtained indicated that increased source strength had an effect on the progression of the senescence phase, particularly the timing of initiation of this stage. Figure 1 presents some of the data from these studies. Under the increased source strength condition, the leaf appears to undergo senescence much earlier compared to normal leaves. Photosynthetic rate decline associated with senescence begins around day 12 in tobacco grown under ambient CO2 levels. A similar loss of photosynthetic rate occurs at least seven days prior in the elevated COj-grown plants. Loss of chlorophyll is also seen to occur much earlier in these plants. All the data obtained suggests an earlier onset of senescence. The decreased source strength condition was obtained using a transgenic tobacco line containing an antisense construct to the small subunit of Rubisco (Miller et al., 2000). The generation of these mutants produced a series of plants with varying degrees of decreased expression of Rubisco protein (Rodermel et al., 1968). The lines with the most severe Rubisco repression resulted in a decrease in photosynthetic rate (Quick et al., 1991). This also corresponded with a decrease in carbohydrate accumulation, including free hexoses (Quick et al., 1991b). This provides us with a decreased source strength condition. The experiments carried out were basically the same as those described in the increased source strength experiments. While elevated COs-grown plants displayed an eariier onset of senescence, antisense plants also exhibited an effect on senescence timing. Decreased source strength however, resulted in a prolongation of the senescence phase. Once again examining 129 the data illustrated in Hgure 1, wild type tobacco leaves have essentially ceased photosynthesizing at day 40, while antisense leaves continue to maintain positive rates IS days further. Chlorophyll amounts are maintained in a similar fashion, well after wild type levels have reached zero, indicative of senescing but longer lived leaves. Increased and decreased source strength conditions appear to have opposing effects on senescence. In one case, senescence initiates earlier than normal, leading to earlier organ death. In the other, senescence may initiate normally but is prolonged and doesn't reach completion until a much longer time, leading to later organ death. As we have demonstrated, source strength does have an effect on leaf development, particularly the senescence phase, and this effect mimics what might be expected by a hexokinase-mediated signaling pathway (i.e., increased sugars leads to down-regulation of photosynthesis and photosynthetic gene expression). In order to prove this however, further experimentation is necessary to confirm sugar-signaling efTects on development The purpose of the third portion of our project was to gain insight into the molecular mechanisms governing carbohydrate influence over leaf developntent. We have already provided evidence of a sugar-regulated model for control over leaf senescence through the completed source strength experiments. The third and fourth portions of this project involve investigating the nature of the mechanism by which this control is exerted in Arabidopsis. Hexokinase signaling has been demonstrated to have such a role. Therefore investigation into hexokinase signaling effects on leaf senescence was the next logical step to testing our hypothesis. There are numerous examples of carbohydrate control over biochemical pathways, and this control can exist on a developmental time scale. It has also been shown that a common mode of action for carbohydrate regulation is through specific changes in 130 sugar-responsive genes, such as the ones involved in photosynthesis. There is also strong evidence that sugars can have an effect on the progression on senescence (Miller et al., 1997). Furthermore, it has been demonstrated that a signal transduction pathway requiring sugar phosphorylation by hexokinase can sense sugars and affect changes in photosynthetic gene expression. Although there have been other sugar sensing systems suggested in higher plants (Smeekens and Rook, 1997), the hexokinase signaling pathway appears to have consequences most similar to senescence. Also, it has been previously suggested that hexokinase may play a role in development (Jang and Sheen, 1994; Jang et al., 1997). Taken together, these points suggest that changes observed during the senescence phase of development may be the result of changes in carbohydrate status sensed through hexokinase signaling. Therefore, we undertook a series of experiments designed to provide the background information on wild type plants to allow us to begin to test the hypothesis of carbohydrate control of leaf senescence via hexokinase signaling, as well as generating the mutants necessary for this study. In some cases, sugar modulation of gene expression was found to be reversible (Krapp and Stitt, 1994). However, similar to ethylene responsiveness, these observations may be due to the developmental stage of the leaf that was examined when changes in carbohydrate export rate were reversed. Transgenic tobacco studies have implied that gene expression changes to sugars may also be permanent (Stitt and Schulze, 1994). Other studies have shown that recovery of elevated COj photosynthetic decline by returning the plants to ambient COj concentrations was not possible after a certain point (Xu et al., 1994). When considering the effects of sugar regulation on photosynthesis on a developmental scale, there 131 may be other factors that can contribute to distinguish short-term fluctuations and long-term permanent changes. We first needed to understand bow leaf development progressed in our new model organism, Arabidopsis thcdiana. This plant was chosen for several reasons, including ease of transformation, rapid life cycle, and experimental precedence. A cDNA of the hexokinase 1 (HXKl) gene was also readily available to us, making Arabidopsis an attractive choice for genetic manipulations. Rnally, an expected senescence progression for Arabidopsis has been determined and delineated (Lohman et al., 1994). This provided us with a previous example for comparative purposes. Another benefit to using Arabidopsis as a model organism is that there have been several genes cloned that show differential expression during the senescence phase (Lohman et al., 1994). These senescence-associated genes, or SAGs, are non- photosynthetic, and can serve as another marker to possible changes in senescence development between plants. Our next set of experiments were designed to create a profile of wild type leaf development that could be used to investigate how certain parameters associated with senescence change in relation to changes in possible sugar signaling factors such as hexokinase activity, invertase activity, and carbon partitioning. Interestingly, we did not observe a pattern similar to tobacco; there was no increase to a maximum before a subsequent decline. Total soluble protein concentrations, pigment contents and photosynthetic rates all decreased over the entire measurement period, including early on before full leaf expansion. However, this observation may be simply due to the increase not occurring during the range of our measurement period. 132 To begin to explore the role of hexokinase signaling in leaf senescence compared to wild type leaves, we generated transformants with increased levels of the enzyme, and then examined changes in various developmental parameters. As stated eariier, mutants with altered hexokinase levels have already been created in Arahidopsis, however the resulting experiments were focused primarily on short-term responses to artificial carbohydrate levels in the ecotype Landsberg (Jang et al., 1997). For this project, our focus was on how increasing hexokinase alters photosynthesis and development throughout leaf ontogeny under normal physiological conditions. The ATHXKl gene in the sense orientation (Jang et al., 1997) was fused to the leaf-specific cab3 promoter. Using this promoter will hopefully provide useful information regarding the location of signal generation in relation to the location of the response. An Agrobacterium-vaeiiiAttA vacuum infiltration method was employed for transformation. Subsequent transformants were screened to isolate isogenic lines with demonstrated alterations in hexokinase for further analysis. Overexpression transgenics were screened with hexokinase assays, looking for progeny with increased activities over wild type levels. Putative antisense mutant seeds were sown on high glucose (6%) MS plates (Jang et al., 1997) to screen for sugar-signaling insensitive plants. RNA analysis confirmed changes in hexokinase message. PCR and Southern analyses were used to isolate homozygous lines from each transgenic type. We now have single-insert mutants from both with demonstrated changes in hexokinase activity. These lines will be used in future analyses. Similar to the tobacco source strength experiments, we will focus primarily on changes in leaf development that indicate the progression of leaf senescence, such as changes in photosynthetic rate, protein, and chlorophyll. These data will be compared to our wild type profile to determine 133 if changes in hexokinase activities are indeed involved in modulating the source strength effects observed on leaf development Future Research Directioiu There are other projects that can effectively use these mutants to explore sugar signaling in higher plants. Based on our cunent understanding of natural carbohydrate metabolism in the leaf, there are potentially two major sources of free hexoses that can serve as substrates for hexokinase signaling. These are breakdown of starch (Schleucher et al., 1996; Weber et al., 2000) and cleavage of sucrose by invertase or sucrose synthase (Foyer et al., 1988). There are mutants of Arabidopsis that accumulate starch because of their inability to transport breakdown products out of the chloroplast. Crossing these plants with the hexokinase mutants may elucidate the possible role of starch breakdown as a signal source. Similarly, the Rodermel lab is working on generating antisense invertase mutants, which could be used for the same purpose. Also, there are many factors that can affect leaf senescence besides carbohydrate metabolism. Among these are light, age and plant growth regulators (hormones). Cytokinins and ethylene are two of the most important hormones involved in senescence regulation (Smart, 1994). However, their mode of action remains unclear. There is evidence that sugar signaling and hormones interact, and the former may override the latter. Analysis of the expression of hormone-related genes (such as biosynthetic enzymes) in the hexokinase mutants would prove interesting. The presence of various hormone response mutants for crosses with the hexokinase mutants could also prove to be valuable. Hnally, there are many genes that are known to be sugar responsive, such as the "feast/famine" genes involved in carbohydrate storage and utilization (Koch, 1996). 134 However, there are many more genes unrelated to carbohydrate metabolism that also exhibit responsiveness. Further characterization of the hexokinase mutants may reveal insights into the role of carbohydrate signaling on these systems. References Foyer CH (1968) Feedback inhibition of photosynthesis through source-sink regulation in leaves. Plant Physiol Biochem 26(4): 483-492 Ham C, Khayat E, and Daie J (1993) Expression dynamics of genes encoding key carbon metabolism enzymes during sink to source transition of developing leaves. Plant Cell Physiol 34:1045-10S3 Jang J-C, and Sheen J (1994) Sugar sensing in higher plants. Plant Cell 6s 1665-1679 Jang J-C, Le6n P, Zhou L, and Sheen J (1997) Hexokinase as a sugar sensor in higher plants. Plant Cell 9:5-19 Koch KE (1996) Carbohydrate-modulated gene expression in plants. Annu Rev Plant Physiol Plant Mol Biol 47:509-540 Krapp A, and Stitt M (1994) Influence of high-carbohydrate content on the activity of plastidic and cytosolic isoenzyme pairs in photosynthetic tissues. Plant Cell Environ 17:861-866 Lohman KN, Gan S, Manorama CJ, and Amasino RM (1994) Molecular analysis of natural leaf senescence in Arabidopsis thaliana. Physiol Plant 92:322-328 Miller A, Tsai C-H, Hemphill D, Endres M, Rodermel S, and Spalding M (1997) Elevated CO2 effects during leaf ontogeny: A new perspective on acclimation. Plant Physiol 115: 1195-1200 135 Miller A, Schlagnhaufer C, Spalding M, and Rodermel S (2000) Carbohydrate regulation of leaf development: Prolongation of senescence in Rubisco antisense mutants of tobacco. Pbotosyn Res 63:1-8 Nie G, Hendrix DL, Webber AN, Kimball BA, and Long SP (1995) Increased accumulation of carbohydrates and decreased photosynthetic gene transcript levels in wheat grown at an elevated CO^ concentration in the fleid. Plant Physiol 108:973-9S3 Quick WP, Schurr U, Scheibe R, Schulze E-D, Rodermel SR, Bogorad L, and Stitt M (1991) Decreased ribulose-1,5-bisphosphate carboxylase-oxygenase in transgenic tobacco transformed with 'antisense' rbcS. 1. Impact on photosynthesis in ambient growth conditions. Planta 183:542-554 Quick WP, Schurr U, Hchtner K, Schulze E-D, Rodermel SR, Bogorad L, and Stitt M (1991b) The impact of decreased Rubisco on photosynthesis, growth, allocation and storage in tobacco plants which have been transformed with an antisense rbcS. Plant J 1: 51-58 Rodermel SR, Abbott MS, and Bogorad L( 1968) Nuclear-organelle interactions: Nuclear antisense gene inhibits ribulose bisphosphate carboxylase enzyme levels in transformed tobacco plants. Cell 55:673-681 Schleucher J, Vanderveer PJ, and Sharkey TD (1996) Export of carbon from chloroplasts at night. Plant Physiol 118:1439-1445 Smart CM (1994) Gene expression during leaf senescence. New Phytol 126:419-448 Smeekens S, and Rook R (1997) Sugar sensing and sugar-mediated signal transduction in plants. Plant Physiol IIS: 7-13 136 Stitt M (1991) Rising CO, levels and their potential significance for carbon flow in photosynthetic cells. Plant Cell Environ 14:741 762 Stitt M, and Schuize E-D (1994) Plant Growth, storage, and resource allocation: from flux control in a metabolic chain to the whole-plant level. In Flux control in biological systems, E-D Schuize ed (Academic Press, San Diego), pp. S7-118 Van Oosten J-J, VTilkins D, and Besford RT (1994) Regulation of the expression of photosynthetic nuclear genes by high CO, is mimicked by carbohydrates: A mechanism for the acclimation of photosynthesis to high COj? Plant Cell Environ 17:913-923 Van Oosten J-J, and Besford RT (1995) Some relationships between the gas exchange, biochemistry and molecular biology of photosynthesis during leaf development of tomato plants after transfer to different carbon dioxide concentrations. Plant Cell Environ 18:12S3-1266 Weber A, Servaites JC, Geiger DR, Kofler H, Hille D, Grtiner F, Hebbeker U, and FlUgge U1 (20(X)) Identification, purification, and molecular cloning of a putative plastidic glucose translocator. Plant Cell 12:787-801 Xu D, Gifford RM, and Chow WS (1994) Photosynthetic acclimation in pea and soybean to high atmospheric CO, partial pressure. Plant Physiol 106:661-671 137 g m m 4M JM 2M § t« IM Time (days) Figure I. Comparison of changes in photosynthetic rate (A and C) and chlorophyll content (B and D) over development between wild type and elevated C02-grown tobacco (in red), and wild type and rbcS antisense mutants of tobacco (in green). 138 ACKNOWLEDGEMENTS There are many individuals that are responsible for helping me to accomplish the completion of my degree, and they deserve thanks. First, 1 would like to thank those who have helped me in my research and education. All of the members of the Rodermel lab, past and present, have made an impact on me and have been invaluable in teaching me about research and science. I would like to especially thank Dr. Carolyn Wetzel for her advice and expertise, Dr. Meng Chen and Dr. Dongying Wu for blazing the trail, and all those who have woiked on this project and put up with me in the process. Special thanks go to my mentors: Dr. Martin Spalding, and my major professor Dr. Steven Rodermel. It was not always easy being a student working for both a biochemist and a molecular biologist, but in the end I have benefited tremendously from their guidance. It was truly a gift to work with them, and learn from them. I would also like to thank Claudia Lemper and Dr. Bernard White, who helped me develop my educating abilities and nurtured my love of teaching. It was a great experience working with them. To my friends who have been there to listen and to give when I needed it, I thank them, and I hope that 1 can be just as important in their lives as they are to me. I would also like to take this opportunity to thank my family for their undying love and support through this time of my life. I have been blessed to have such caring and devoted family members that have contributed to my accomplishments much more than they sometimes realize. My parents and my grandfather have provided me with all that I could need and want, and this degree is yet another success that is due in large part to their love and influence. 139 Finally, 1 want to thank my wife Danelle, who is more important to me than any degree I could ever achieve. Her love and support have been essential to me, and this moment would truly not be the same without sharing it with her completely. I thank her for everything she is to me and for making me happy.