Sponsored by the Department of Energy, Office of Science,
Program for Ecosystem Research ( )

Introduction Overall
Model System Justification
and Description
Hypotheses Conceptual
Relevance to
DOE Mission


We propose to modify nitrate reductase (NR) activity in Arabidopsis and follow the consequences of this modification through multiple levels of biological organization; cells to ecosystem. An overarching hypothesis of this project is that a single-enzyme change affecting an important biological process (i.e. nitrate assimilation) will translate across multiple levels of biological organization to produce detectable and predictable responses at the ecosystem level. A combination of hydroponic and soil-based mesocosms will allow us to establish mechanistic links between adjacent levels using tools of molecular biology, genomics, biochemistry, physiology, population genetics, microbiology, and ecology. Hydroponic systems will allow the source and concentration of nitrogen to be closely controlled. A soil-based system will also be used to address how mechanistic relationships at cellular and organismal levels influence population, community, and ecosystem-scale processes. A factorial study will involve wild type (WT) and transformed Arabidopsis, and mixtures of the two, exposed to ambient and elevated [CO2]. Elevated [CO2] treatments will help identify causal associations among levels of biological organization by accentuating interactions between the carbon and nitrogen cycles. As a secondary goal, we expect that this research will contribute to fundamental understanding of the linkages between NR activity and photosynthesis, and more generally to the importance of N metabolism in determining plant responses to elevated [CO2]. Results from these investigations will contribute to a more mechanistic description of how environmental change influences structure and function of terrestrial ecosystems; a primary goal of the DOE Program for Ecosystem Research.

Introduction [ Top ]

"…each level [of biological integration] offers unique problems and insights, and…each level finds its explanations of mechanisms in the levels below, and its significance in the levels above." – George A. Bartholomew, 1964

Ecological systems are characterized by highly complex and non-linear interactions among a large number of individual elements. These elements are organized along hierarchical levels, with lower levels represented by cells and macromolecules and higher levels by ecosystems. In between, intermediate levels of biological organization (i.e., tissues, organism, and communities) impose constraints and boundary conditions on adjacent levels. Such complexity leads to emergent properties and unexpected dynamics, complicating our ability to understand and predict system behavior. Understanding how changes at one level of biological organization will alter emergent patterns or mechanisms at another level of biological organization is one of the most pressing problems in ecology.

Biological and ecological systems are characteristically hierarchical and there is a constant interplay between events at different levels. This interplay extends from the events that happen very slowly at the scale of ecosystems down to the most rapid events operating at the scale of cells and macromolecules. A unique molecular event, like a single-gene mutation, could be amplified to the extent that it changes the function of an entire ecosystem. Unfortunately, biology and ecology have traditionally separated along these same lines of organization, with overlap occurring at the level of the organism (Figure 1). Such separation impairs understanding how effects integrate across levels of biological organization.

Figure 1

Overall Objective [ Top ]

In this proposal we address the question of how these different levels of organization can be integrated, or more specifically how mechanisms expressed at one level of organization can be understood in terms of processes operating at a different level of organization. Our approach centers on modifying nitrate reductase activity (NR) in Arabidopsis and following the consequences of this modification through multiple levels of biological organization; cells to ecosystem. To further deconvolute translation of the effects of the NR modification, we will perturb the mesocosm by altering the [CO2], which is known to produce detectable effects on plant function that are intimately associated with N-status of the plant. An overarching goal of this project is to demonstrate that a single-enzyme change affecting a fundamental biological process can translate across multiple levels of biological organization to produce detectable and predictable responses at the ecosystem level. To this end, we are manipulating NR and [CO2] under highly simplified conditions in order to expose linkages between macromolecules (transcripts and metabolites) and organs (roots and leaves); organs and organisms (the plant); organisms and populations; populations and communities (microbial and herbivore); and between communities and a highly simplified ecosystem (the mesocosm). As a secondary goal, we expect that this research will contribute to fundamental understanding of the linkages between NR activity and photosynthesis, and more generally to the importance of N metabolism in determining plant responses to elevated [CO2]. These goals will be achieved with coordinated measurements using the tools of molecular biology, genomics, metabolomics, biochemistry, physiology, population biology, microbiology, and ecology.

Model System Justification and Description [ Top ]

Linkages among hierarchical levels of biological organization can be most effectively explored using a highly simplified system with a tightly controlled perturbation at the macromolecular level. This allows isolation of macromolecular effects and mechanistic interpretations of the translation of effects across hierarchical levels. We therefore propose the new Hierarchical Experimental Responses at Macromolecular to Ecosystem Scales (HERMES) research project. This new project will be used to explore linkages among levels in simple mesocosms containing Arabidopsis, focusing initially on a targeted disruption of a single enzymatic process, the conversion of nitrate to nitrite by NR. Arabidopsis is widely recognized as the premier model plant in molecular biology, and the same characteristics that make Arabidopsis an excellent model species for genetic studies will also facilitate successful exploration of hierarchical scales of organization. These characteristics include: 1) small size, to facilitate construction of highly replicated mesocosms with minimal edge artifacts; 2) short generation time, to enable exploration of effects on individual fitness and translation to population-level effects; 3) self-pollination, which results in genetically uniform populations and isolation of single-enzyme effects; and 4) ease of transformation, which will allow rapid production of mutants with a range of nitrate reductase activities. In addition, the enormous body of literature on Arabidopsis biology and the well-developed molecular and biochemical tools and protocols will allow dissection of mechanistic effects of experimental manipulations at each hierarchical level with unparalleled precision.

Conviron growth chambers

The hydroponic experiments will be housed in new Conviron growth chambers.

ORNL greenhouses

The mesocosm facilities will be housed within greenhouses that were recently constructed at ORNL.

Hypotheses [ Top ]

The following hypotheses describe how information initiated by a targeted change in NRactivity can be translated across levels of biological organization from macromolecules to the whole mesocosm. The hypotheses reflect a plausible sequence of events, beginning with biochemical changes in the NR- plants that increase nitrate concentrations in shoots, giving rise to changes in carbon allocation to roots and shoots, and ultimately leading to changes in population structure and ecosystem function. In addition, the hypotheses address how elevated [CO2] can modulate this sequence of events as a result of links between carbon acquisition and nitrogen supply/metabolism and/or interactions between plants and changing functional composition of microbial communities.

Hyp. 1: Reduced nitrate reductase activity will cause increases in leaf nitrate concentration, which will affect multiple biochemical pathways, including negative feedbacks on starch synthesis and positive feedbacks on organic acid synthesis via the glycolytic and TCA cycle pathways, and increased expression of N assimilation genes.

Hyp. 2: Leaf nitrate concentration regulates S/R ratio by controlling carbon partitioning to organic acid synthesis and carbon allocation between plant organs, limiting the availability of soluble carbohydrates to roots.

Hyp. 3: Elevated [CO2] will reduce nitrate assimilation in WT plants but have no effect on nitrate assimilation in NR deficient plants.

Hyp. 4: Impaired nitrate assimilation and altered root:shoot ratio will translate to changes in population structure over time mediated by competitive exclusion of mutants by WT plants. This effect will be exacerbated in elevated [CO2].

Hyp. 5: Reduced C inputs into the rhizosphere of the NR- plants will alter the relative abundance, composition and functional capacity of the associated rhizosphere bacterial community, further limiting plant growth and the ability of the NR- plants to compete in mixed mesocosms.

Hyp. 6: The increased reliance of NR- plants on NH4-N will lead to increased competition with autotrophic nitrifying bacteria for available soil ammonium, further limiting success of the NR- phenotype through a negative feedback from the soil bacterial community that alters soil N status and plant performance.

Hyp. 7: Net ecosystem exchange (NEE) – a measure of total system productivity – will decrease over time in the pure NR- mesocosm, increase over time in the NR--WT mixture, and remain constant in the WT mesocosm. These effects will be mediated by the population-level consequences of competition and the community-level effects of increases in nitrifying bacteria.

Hyp. 8: Elevated [CO2] will result in net increases in NEE in all mesocosms, but the effects will be inversely proportional to the abundance of NR- plants in the system, and there will be a negative interaction between NR- plants and nitrifying bacteria.

Conceptual Approach [ Top ]

The research described herein will explore linkages among levels of biological organization in two complementary systems of plant culture, or mesocosms. We will focus on the propagation of treatment effects involving the plant NR gene and atmospheric [CO2] across levels of a hierarchy encompassing Arabidopsis plants. We will follow the effects of these manipulations through the cell, organ, and organism levels primarily using hydroponic experiments, which will allow intensive physiological and biochemical characterization under highly controlled conditions. Growth of Arabidopsis in hydroponic systems will allow the source and concentration of nitrogen supply to be closely monitored, and provide a system from which uniform root and shoot material can be harvested for genomic, biochemical, and physiological characterization up to the level of individual plants. Furthermore, because of their small size and the short life cycle of Arabidopsis, many different experiments can be run during the course of this project.

A soil-based system will also be used to address how mechanistic relationships at organismal levels influence population, community, and ecosystem-scale processes. These systems will be larger than the hydroponic mesocosms, thus facilitating study of long-term dynamics of seed dispersal, germination, and plant competition over multiple life cycles. We will determine the applicability of the hydroponic experiments (and translation across the levels of organization below the individual) to the soil-based experiments by repeating all measurements taken at the individual level in both systems (Figure 2). We will assess translation of effects from the individual plant to the population level by sowing an equal mixture of NR- and WT plants and following the trajectory of population development over successive generations in the mesocosms. Comparison of mesocosms containing pure NR- or WT plants to mesocosms with mixtures will provide a framework for assessing translation of treatment effects from the population to the community and ecosystem levels. Community effects will be assessed by examining the composition and function of microbial and herbivore communities over time as organic matter accumulates in the system and changes the fundamental biotic composition of the soil. Finally, we will assess translation of treatment effects from the community to ecosystem level by assessing whole-system properties including net ecosystem exchange, and nitrogen leaching.

Relevance to DOE Mission [ Top ]

Our aim in this proposal is to deepen scientific understanding of how changes in C and N metabolism at one level of biological organization translate to changes at the ecosystem level. Ecosystems sense and respond to elevated [CO2] through plants. Most of the effects of elevated [CO2] on plants and their ecosystems stem from the increase in photosynthetic CO2 uptake. In the long-term, N supply is a critical factor determining whether there will be a sustained and maximal stimulation of plant and ecosystem productivity by rising [CO2]. Therefore, understanding the processes associated with the acquisition and subsequent metabolism of nitrogen (NO3-) and carbon (CO2) will lead to an improved understanding of how terrestrial ecosystems respond to elevated [CO2]. Furthermore, increased knowledge of the translation of material and information associated with carbon and nitrogen metabolism between levels of biological organization will lead to increased mechanistic understanding of many other important plant and ecosystem processes. For example, increased nitrogen use efficiency resulting from photosynthetic acclimation to elevated [CO2] may affect species composition in nutrient poor ecosystems. The C:N ratio of leaf litter may influence microbial decomposition rates which, in turn, may lead to ecosystem level feedbacks to biogeochemical cycles. Increased foliar sugar content and decreased foliar protein content may increase susceptibility to insect herbivory in plants grown in elevated [CO2]. This approach of focusing on the interaction between nitrogen and carbon metabolism directly supports the overall objective of the PER: to measurably improve the scientific basis for predicting or detecting effects of environmental changes associated with energy production on terrestrial ecosystems and their component organisms and processes.

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January 13, 2005