STATEMENT OF THE ISSUES AND JUSTIFICATION

Necessity of Photosynthesis Research

Photosynthesis is essential to life on earth as it converts sunlight into biochemical energy used by nearly all life forms. It is the primary process for generating plant biomass. As a result of photosynthesis, carbon dioxide, as well as inorganic nitrogen and sulfur, are converted into organic molecules (e.g., sugars, lipids, amino acids and other cell building blocks). Oxygen is generated, as a byproduct, through photosynthetic water oxidation. Thus, plant and algal photosynthesis affects global geochemical processes, in particular carbon cycling (29), and is an important factor to be considered in global climate change models. Aside from these fundamental aspects of photosynthesis, agricultural production of food, feed, fiber, natural chemicals, and biofuel feedstocks is directly affected by limitations in the rate and yield of photosynthesis and the capacity to utilize fixed carbon (118, 132). Some of the greatest challenges that confront humankind – feeding an exponentially growing global population, supplying sufficient energy to sustain this global population, and averting negative environmental impacts due to human activities – can or need to be addressed using photosynthetic organisms (28). Therefore, the need for state-of-the-art photosynthesis research to improve the efficiency and productivity of this process in traditional crops or for the development of novel crops and products has never been more urgent.

Collaborators participating in this regional project will place considerable focus on understanding and improving the response of photosynthesis to genetic, developmental and environmental factors that limit productivity. The research spans all levels of organization from the genetic, molecular and cellular through the leaf, whole plant, and canopy.  Particular emphasis will be placed on abiotic stresses (i.e., heat, cold, drought and salinity), nitrogen- and water-use efficiency, carbon flux pathways, and the signal transduction mechanisms that initiate plant responses, because these are critical to developing a climate-smart agriculture (100). Factors that enhance or limit agricultural productivity generally do so by impacting photosynthesis.  The gains in yield of the major crops over the past half-century have come about primarily from breeding for greater harvest index (HI) and through management, particularly the application of fertilizer (39).  For well-fertilized crops, HI has approached the maximum achievable for many crops, and future yield gains will depend on increasing total production (113).  Greater productivity, which is necessary to meet the food, feed, fuel and fiber needs of a growing world population (102), requires improving the basic efficiency of the photosynthetic process for light energy capture, and the conversion of this energy to chemical form for the synthesis and utilization of organic molecules (113).  For maximum benefit, the efficiency of photosynthesis must also be resilient to changes in temperature, CO₂, and precipitation / drought anticipated from climate change.

Importance and Consequences

Global climatic trends are negatively impacting the yields of major crops used for the production of food, feed, fiber, and biofuels (92). Sustaining the productivity of traditional crops will require improvements in the efficiency of photosynthesis that go beyond traditional breeding and selection (93). Similarly, the development of novel feedstocks for biofuel/chemicals generation in a way that is sustainable, commercially-viable, not in competition with food production, and that mitigates greenhouse gas emissions requires novel approaches and non-traditional crops including algae (109, 123) and cyanobacteria (30). Only a concerted and vertically-integrated effort encompassing all aspects of photosynthesis will ensure that appropriate solutions can be found for some of these most pressing problems currently facing society. Innovative thinking will be required that does not stop at traditional agricultural systems and crops, but may enable transitioning to new systems dedicated to the production of biofuels and chemicals instead of food. As photosynthesis is at the basis of biomass production, we need to find innovative ways to overcome its limitations. Failure to do so now will limit our future ability to produce sufficient food, feed, fiber, and fuel in a rapidly changing climate.

Technical Feasibility

Genomic resources and next generation sequencing and transformation technologies have advanced to the point that a wealth of information can be generated for any species of plant or alga (e.g., 10,000 plant genomes project) (116), in a relative short time span and at reasonable costs. Thus, the raw material for the genetic manipulation of novel crops or algal strains is readily available. Large-scale phenomics focused on chloroplast proteins in multiple plant species including genetic models such as Arabidopsis (24) and crops (127). Moreover, using comparative genomics, reconstruction of metabolism from gene expression and genomic data has become readily feasible for any organism. One example is the metabolic reconstruction of the alga Chlamydomonas reinhardtii (53) and the cyanobacterium Synechocystis sp PCC 6803 (64). In addition, our knowledge and analysis of primary metabolism of photosynthesis in plants and associated metabolic networks has advanced to levels (71) that can enable the rational design of novel crops, for example, establishing crassulacean acid metabolism (CAM) in plants with C₃ metabolism (129). Despite this progress, the task of genetically transforming crop plants and analyzing the resulting phenotypes is still tedious and time consuming. Synthetic biology efforts with plants, involving stacking or replacing multiple genes, are lagging behind those for bacterial systems (97). Moreover, photosynthesis is one of the most complex processes found in nature, requiring hundreds of genes and proteins, and multiple and overlapping levels of regulation. Thus, to enable rapid progress in basic discovery and to devise strategies for the improvement of photosynthesis in crops, facile model organisms such as Arabidopsis (110) or model microalgae and cyanobacteria such as Chlamydomonas (98) and Synechocystis (20, 117) will have to be employed to quickly identify the most promising directions for photosynthetic pathway and crop productivity improvement. Following this guidance, collaboration with geneticists and breeders, associating traits with genomic regions of mapping populations or diversity panels can support marker-assisted selection (22, 41). Such an integrated approach requires multifaceted expertise and, thus, will benefit from synergy derived from a multi-state investigator effort.

Multi State Effort

Providing a conceptual framework through the current project, this group of scientists, bringing together a complementary set of expertise, and others located throughout the US have already successfully made progress on understanding diverse aspects of photosynthesis. While global issues as laid out above are addressed, practical solutions to these problems often have local solutions (e.g., by taking into account climatic zones to which specific crop species or algae are adapted). Continued effort by the current group will contribute towards these main goals while also enabling local solutions of particular value to the participating states. Partners in this endeavor are listed for each focus area below.

Likely Impacts

Efforts by the group are organized into four themes (Objectives). While the details and outcomes will be discussed below in the main body of the proposal, likely impacts falling under these themes can be briefly summarized as follows:

1. Photosynthetic Capture and Photorespiratory Release of CO₂. Photosynthetic carbon fixation and photorespiratory release of CO₂ have long been recognized as limitations to crop productivity (37, 94, 99, 111). Carbon metabolism in photosynthesizing leaves involves the Calvin-Benson cycle but also other carbon fluxes. A more complete understanding of these carbon-reaction processes will identify mechanisms leading to improved photosynthetic efficiency. The carboxylating and oxygenating enzyme, Rubisco, is central to these efforts. As temperature increases, photorespiratory pressure in C₃ plants increases due to decreased Rubisco specificity for CO₂ relative to O₂. However, the carboxylating activity of Rubisco is greater in C₄ and CAM photosynthetic pathways, relative to that in C₃ photosynthesis, in spite of the warmer ecozones these occupy. Understanding the gene expression regulation and regulatory network of C₄ and CAM metabolic activities is the key to fully elucidate the evolutionary history of C₄ and CAM photosynthesis, and to take advantage of these for improved crop productivity. The rates of photosynthesis in C₃, C₄ and CAM plants can be limited by the availability of CO₂ at the initial site of carboxylation.  Understanding how leaf biochemical and anatomical traits influence the concentration of CO₂ at the site of carboxylation may guide strategies to increase photosynthetic rates. Studies will utilize genomic analysis, loss of function mutants, ¹³CO₂ isotope studies and metabolite analysis to understand carbon fluxes involved in photorespiration, transcriptional regulation of C₄ and CAM metabolism and CO₂ availability at the initial site of carboxylation. Likely impacts include an instrumental knowledge of factors regulating carbon fluxes in light-independent processes including effective CO₂ concentrations at carboxylation sites, the costs and benefits of ancillary carbon paths in photosynthesizing cells, and circadian regulation in C₃, C₄ and CAM photosynthesis, with application to crop improvement (MI-ABR, NV-AES, TX-AES, WA-AES).


2. Strategies to optimize the assembly and function of the photosynthetic membrane. Chloroplasts are the organelles that perform photosynthesis in both plants and algae. Chloroplasts also contain a large number of enzymes, highlighting the role of this organelle as a primary biochemical production factory. As semi-autonomous organelles, chloroplasts do not function by themselves, but rely on extensive communication with other organelles and compartments within the cell, and with the whole plant. The import of nuclear-encoded proteins or membrane lipids assembled at the endoplasmic reticulum provide two examples of such interactions (66, 114). As primary photosynthate and many other metabolites (e.g., fatty acids and most isoprenoids), are only synthesized in chloroplasts, they have to be exported to be available to other cellular compartments. The integration of chloroplast biogenesis into overall cell development requires intricate signaling processes, as does the adjustment of the photosynthetic electron transport chain to changing conditions. Within the chloroplast, the capture and conversion of light energy by photosynthesis occurs at a specialized structure called the thylakoid membrane, which is itself dynamically remodeled in response to developmental and stress cues. The architectural dynamics of the stacked grana thylakoids are involved in regulating and maintaining photosynthetic Studies will focus on how thylakoid membranes change their shape, the functional consequences of structural alterations, and effects of whole-plant stresses and developmental cues on the thylakoid membrane and chloroplast lipid changes. Likely impacts are a better understanding of photosynthetic energy transduction and transformation, the development of the thylakoid membranes under developmental and stress regimens, and the development of tools that can be used to assess chloroplast membrane connectivity (MI-ABR, NE-ARD, WA-AES).


3. Mechanisms Regulating Photosynthate Partitioning. Manipulation of carbon partitioning and understanding its regulation is central to advances in yield and heterologous product formation in cereal, oilseed crops, and algae. This effort will contribute with the design of new photosynthetic systems for specialty and commodity chemicals production that possess enhanced carbon flux to innate or new sinks, e.g., starch, useful triacylglycerols (27), and antioxidant or isoprenoid products (81). Several related efforts are expected to have positive impacts on plant growth rates and yield. The “starch partitioning” project, comprising the mapping of genes impacting leaf starch levels will use field and greenhouse studies to determine the impact of sink capacity on photosynthetic rates and plant productivity. This will lead to information on mechanisms regulating photosynthate partitioning toward starch biosynthesis / accumulation. Another approach seeks to evaluate the function of PHO1, a higher plant plastid-localized phosphorylase in starch biosynthesis and its newly discovered interaction with photosystem I. The role of PHO1 in starch partitioning and photosynthesis will be investigated in relation to enhanced plant growth rates and grain yields. The “lipid partitioning” project will investigate plants with altered metabolism to accumulate significant storage reserves, such as lipids, in leaves. Metabolic studies will assess the functional and productivity consequences of photosynthetic carbon partitioning to lipid and storage in leaves, converting plant leaves into lipid storage tissues. The “antioxidants enhancement project” investigates evolutionary aspects of leaf antioxidant content in relation to biomass accumulation. Small antioxidant amounts, applied exogenously to plants, improve photosynthetic efficiency by altering the partitioning of light to photochemistry or non-photochemical quenching. This project will investigate the underlying reasons and explore the possibility of improving antioxidant pools in vivo. The “isoprenoids partitioning” project aims to enhance flux toward the synthesis of plant essential oils and related compounds with applications in flavor, fragrance, synthetic chemistry, and biopharmaceuticals. All of the above projects would benefit from the “fusion constructs” project, which will deliver a platform for substantially enhanced carbon partitioning toward a target biosynthetic pathway, via pathway enzyme over-expression (117). This would alleviate rate- and yield-limiting catalytic steps in the generation of endogenous and heterologous compounds.  Likely impacts include definition of the mechanisms that regulate photosynthate partitioning into the biosynthetic pathways for sucrose, starch, lipids, antioxidants, and isoprenoids. The effect of innate or heterologous sink strength on rates and capacity of photosynthesis will be assessed in each case. The above comprise an integrated approach to understanding carbon partitioning and its effect on product synthesis and accumulation.  (CA-AES, MO-ARS, MT-AES, NE-ARD, WA-AES).


4. Developmental and Environmental Limitations to Photosynthetic Productivity. Factors such as leaf anatomy (112) or environmental stress conditions, such as high light (86), excess salinity, phosphorous deficiency, drought or heat stress (105) greatly affect photosynthesis and plant productivity. Moreover, leaf stomata, affecting photosynthetic productivity as a ‘gateway’ for CO₂ influx, are subject to complex active regulation, responsive to factors including light intensity, temperature, vapor pressure, and leaf CO₂ partial pressure. Heat stress during flowering and grain filling alters the flux of assimilates in wheat, corn, and sorghum (54, 95). Included are pollen infertility, which reduces grain number, while diminished assimilate flow reduces grain weight. Genomic analysis in this objective seeks to identify critical loci encoding heat and water stress tolerance traits. An additional approach combines adaptive traits (CAM, tissue succulence, thick cuticles and epicuticular wax, low stomatal density with high responsiveness and rectifier-like roots) to develop crops that are resilient to severe heat and water-deficit stress. Abiotic stress signaling systems involve inositol pyrophosphates (signal for phosphorus uptake and utilization) and calcium-dependent protein kinases (regulating responses to biotic and abiotic stress, metabolism, vegetative development and sexual reproduction). Small signaling peptides are known to regulate plant development. However, the functions of dehydration-stimulated peptides on water-limited photosynthesis have not been investigated. Studies will utilize genomic analysis, loss of function mutants, and metabolite analysis to identify factors regulating photosynthetic and photosynthate partitioning responses to abiotic stresses such as heat, water deficits, excess salinity and phosphorus deficiencies/toxicity. Activities in Objective 4 will thus be integrated with those of Objective 3 to provide a more holistic perspective on photosynthate partitioning, as this is defined by genetic and environmental stress conditions. Likely impacts include better understanding of the effect of abiotic stress on carbon fluxes, development of genomic tools for crop improvement and novel genetic resources for xeric conditions. (IN-AES, KS-AES, MO-AES, MS-AES, NE-ARD, NV-AES, VA-AES, TX-ARS).