Duration: 10/01/2007 to 09/30/2012

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

Statement of Issues and Justification

Photosynthesis is the primary determinant of crop productivity. It is the single process on earth that converts sunlight into biomass, sequesters atmospheric CO2 into carbohydrates, and liberates O2. Photosynthesis and the formation of food, fiber, and biomass are dramatically limited by environmental, biochemical, and genetic constraints. Alleviation of some or all of these constraints could lead to substantial increases in plant productivity.

New approaches will be required to enhance crop productivity and improve agricultural sustainability. To achieve these goals, it will be essential to gain fundamental knowledge of all aspects of photosynthesis from underlying metabolic components that control assimilate production and utilization, to basic enzymology and cellular infrastructure. This knowledge will include an in-depth understanding of the regulation of important photosynthetic enzymes and how environmental and developmental signals affecting photosynthetic processes are perceived at the molecular and gene levels. Collectively such knowledge will provide novel opportunities for crop improvement.

The proposed multistate research project brings together outstanding, highly productive photosynthesis investigators from across the country in an integrated effort to broaden our understanding of critically important areas of photosynthesis research. We propose a synergistic, cooperative research program that concentrates on four areas of photosynthetic regulation:

1. Plastid function and intracellular communication. The purpose of this research is to identify regulatory pathways by which plastid biogenesis and photosynthetic functions are modulated. Direct signaling pathways and photosynthetic protein transfer between chloroplasts and the nucleus will be studied, as will homeostatic processes that utilize sugar sensing to co-ordinate photosynthetic supply with cellular and developmental programs. The collaborating units include IA-AES, OH-AES, ME-AES, and SC-AES (4 institutions, 4 research labs).

2. Photosynthetic capture and photorespiratory release of CO2. The goal of this research is to determine and modify the biochemical and regulatory factors that impact photosynthetic capture and photorespiratory release of CO2. Particular emphasis will be placed on understanding protein-protein interactions and post-translational modifications of key photosynthetic enzymes involved in primary and secondary CO2 assimilation, as well as the mechanisms that control carbon flux through primary and secondary metabolic pathways. The collaborating units include IL-ARS, KY-AES, MO-AES, NE-AES, and NV-AES (5 institutions, 6 research labs).

3. Mechanisms regulating photosynthate partitioning. The objective of this research is to gain insight into the mechanisms that regulate photosynthate partitioning into pathways of biosynthesis and use of sucrose, starch, and sugar alcohols as well as lipids. These studies will examine interactions between compartments of the cell, between plant parts, and the partitioning of carbohydrates and lipids between transport, storage, and stress-protective functions. The collaborating units include FL-AES, IA-AES, IL-AES, MI-AES, NE-AES, PA-AES, VA-AES, WA-AES (8 institutions, 11 research labs).

4. Developmental and environmental limitations to photosynthesis. The aim of this research is to analyze the limitations and environmental factors that influence photosynthetic productivity at the whole plant and canopy levels. Particular emphasis will be placed on abiotic stresses (temperature, water, and salinity), nitrogen use, and global atmospheric change. This work will integrate understanding developed here and under objectives 1 through 3 to optimize photosynthetic production and yield under current and future environmental conditions. The collaborating units include IA-AES, IL-AES, KS-AES, MI-AES, MS-ARS, NE-AES, NV-AES, WA-AES, VA-AES (9 institutions, 12 research labs).

Related, Current and Previous Work

Objective 1 Plastid Function and Intracellular Communication

The biogenesis of the photosynthetic apparatus and the expression of most photosynthetic genes require co-ordination between the plastid and nucleus. Both anterograde (nucleus-to-chloroplast) and retrograde (plastid-to-nucleus) signals have evolved to facilitate these regulatory interactions. The characterization of plant variegation mutants, which have green and white (or yellow) sectors in normally green organs, has already proven to give novel insights to retrograde signals and to plastid functions. Additionally, cellular homeostasis mechanisms ensure that photosynthetic capacity is responsive to metabolic output and to cellular growth requirements. Prominent among these mechanisms is the role of sugars as signaling molecules. Furthermore, a useful model organism, Elysia chlorotica, is being studied to identify some of these intraorganelle, regulatory mechanisms that occur in at least one path of plastid endosymbiosis.

The immutans and var2 variegation mutants of Arabidopsis arise from sorting of albino and green plastids early during plastid development (Aluru et al., 2006). Both responsible genes have been cloned and characterized. IMMUTANS is thought to protect early developmental formation of thylakoid membranes from photooxidation, while VAR2 is suggested to function in photorepair via D1 protein turnover. Recent suppressor screens by the Rodermel lab (IA-AES) have identified mutants in each variegation background. A var2 suppressor was identified as a stromal chaperone. Further characterization of the suppressor lines will greatly improve our understanding of chloroplast biogenesis.

Plant sugars regulate many aspects of organismal growth and function through both nutritive metabolic effects and gene regulatory mechanisms. In Arabidopsis, glucose can modulate expression of about 1000 genes (Price et al., 2004) to promote seedling development, leaf expansion, vegetative growth, flowering, and senescence (Moore et al., 2003; Xiao et al., 2000). Arabidopsis hexokinase1 (HXK1) is the best characterized plant glucose sensor. HXK1 functions as a moonlighting protein with distinct metabolic and gene regulatory properties, and commonly represses photosynthetic gene expression (Moore et al., 2003). Functional characterization of the HXK gene family has established that two hexokinase-like proteins also have related, key roles in glucose signaling (SC-AES). Furthermore, recent data in the Moore lab (SC-AES) indicate that HXK1 also regulates mitochondrial abundance in mesophyll cells. Using global transcription profiling, the Jang lab (OH-AES) has identified 184 sugar responsive transcription factors in Arabidopsis. One of those acts as a novel promoter of flowering. Further characterization of key targets of glucose signaling will allow us to understand the cellular processes that couple photosynthetic carbohydrate production with the control of plant growth and development.

Understanding the process of endosymbiotic plastid acquisition and retention offers unique insight into the establishment of regulatory processes that coordinate nuclear and plastid functions (Rumpho et al., 2000). Elysia chlorotica is a shell-less, sea slug that acquires plastids upon feeding on the chromophytic alga, Vaucheria litorea. The conferred ability to carry out photosynthesis is essential for development of the juvenile sea slug. In addition, the chloroplasts are retained by the sea slugs for their entire 10-month life-cycle, supporting photosynthesis despite the absence of any algal nuclei. Considering that about 90% of proteins normally required for plant plastid function are nuclear encoded and must be imported from the cytosol, the level of chloroplast activity observed in the animal cell is quite remarkable. The Rumpho lab (ME-AES) has recently identified two partial genes for plant nuclear-encoded photosynthesis proteins, the photosystem II Mn-stabilizing protein (psbO) and the Calvin Cycle enzyme phosphoribulokinase (prk), in the sea slugs DNA. Further identification of DNA elements that have been horizontally transferred to the sea slug is one component to understanding the host cell nuclear contribution to maintaining the engulfed plastids. Another key component is to identify and characterize the chloroplast proteome in the sea slug over the entire life-cycle to better understand the role of the nucleus in chloroplast gene expression and the stability of chloroplast proteins.

Objective 2: Photosynthetic Capture and Photorespiratory Release of CO2

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the fixation of CO2 from the atmosphere and thereby determines the influx of carbon into the Earths biosphere. Collaborative review of the Rubisco field has been presented by NE-AES, AZ-ARS, KY-AES, and IL-ARS (Spreitzer and Salvucci, 2002; Houtz and Portis, 2003). With respect to the genetic engineering of Chlamydomonas Rubisco (NE-AES), substitutions in the small subunit have identified regions that can influence carboxylation catalytic efficiency and CO2/O2 specificity via structural interactions with the active-site large subunit. Based on a bioinformatics approach, differences between Chlamydomonas and crop plant large subunits have been defined that may account for the catalytic diversity of phylogenetically-diverse Rubisco enzymes. Such work has culminated in the co-engineering of large and small subunits to create a Chlamydomonas enzyme that has the higher CO2/O2 specificity characteristic of crop-plant Rubisco (Spreitzer et al., 2005). Regions far from the active site defined by these methods may serve as targets for genetic selection aimed at improving Rubisco. Furthermore, the Spreitzer group (NE-AES), in collaboration with Dr. Inger Anderson (Uppsala, Sweden), now has the highest resolution x-ray crystal structure of all Rubisco enzymes (Taylor et al. 2001). Recently-solved structures of mutant enzymes may provide a rationale for the design of an improved Rubisco (Karkehabadi et al., 2005a, b).

The importance and role of co- and post-translational protein processing in the chloroplast has been established (Dirk et al., 2006). The Houtz group (KY-AES) have demonstrated that chloroplast-localized peptide deformylase (DEF) is essential in all plants, and the lethality of DEF inhibition in plants occurs primarily through the rapid destabilization of newly translated D1 polypeptides  one of the core proteins of the photosystem II apparatus (Hou et al., 2005). This destabilization leads to a disassembly of the photosystem II protein complex and a loss of photosynthetic activity, ultimately resulting in bleaching and necrosis. These results are consistent with in vitro experiments that tested a variety of peptide analogs of select chloroplast-translated proteins as DEF substrates, and found that the D1 polypeptide was the preferred polypeptide substrate. Recent acquisition of a high-resolution crystal structure for DEF has identified key active-site residues essential for activity and instrumental in polypeptide substrate specificity. Post-translational methylation of Lys-14 in the large subunit of Rubisco has been shown to be a consequence of chloroplast-localized Rubisco LSMT, a SET domain protein methyltransferase (Trievel et al., 2003). Recent studies have identified several other chloroplast-localized proteins as substrates for Rubisco LSMT including gamma tocopherol methyltransferase and aldolase. Post-translational methylation of aldolase results in a 2-fold increase in catalytic activity and represents a novel mechanism for influencing the activity of a key chloroplast enzyme.

Rubisco activity is maintained by another chloroplast protein, Rubisco activase. Rubisco activity might be increased to improve plant growth by altering its interaction with Rubisco, which is likely to contribute to the inhibitory effects of moderate heat stress. Past work showed that neither Rubisco nor activase from one species of plants, the Solanaceae, worked well in combination with the other protein from other species. Collaborative work between the Portis and Spreitzer groups (Larson et al., 1997, Ott et al., 2000) showed that this peculiar specificity preference is determined by two amino acids on the surface of Rubisco. During the current project period, collaborative work by Portis and Salvucci (Salvucci and Portis, 2005) identified a substrate recognition region in activase in which two amino acids may directly interact with the two previously identified amino acids in Rubisco.

In C4 and Crassulacean (CAM) plants, phosphoenolpyruvate carboxylase (PEPC) catalyzes the initial fixation of atmospheric CO2 into C4-decarboxylic acids. Because this pathway concentrates CO2 in the vicinity of Rubisco, PEPC and its regulation are attractive targets for the genetic engineering of improvements in CO2 fixation. The Cushman (NV-AES) group has demonstrated that oxidative stress is normally induced during the abiotic stress conditions required for CAM induction (Borland et al., 2006). These findings suggest that CAM may increase the oxidative burden on the plant. However, exogenous exposure of ice plants to ozone, a form of reactive oxygen stress does not fully induce CAM suggesting that the production of ROS alone is insufficient for the induction of CAM. A detailed investigation into the molecular genetic changes present in C3 photosynthesis, weak CAM, and strong CAM plants will help us to define the exact genetic and metabolic requirements for the performance of CAM and to define the mechanisms of CAM evolution.

Objective 3: Mechanisms Regulating Photosynthate Partitioning

Critical steps in the conversion of photosynthate to carbohydrates (sucrose, polyols, starch etc.) and lipids can affect the photosynthetic process itself, as well as the amount of fixed carbohydrates and lipids allocated to yield. Key points in this regulation offer exciting avenues for manipulation; however, the underlying mechanisms that limit carbohydrate and fatty acid synthesis and carbon flux into harvestable organs remain unclear. Feasible long-term targets for altered partitioning begin at the metabolic level (photosynthetic end-products) and extend to the whole plant level (importing plant parts). Recent work from NC-142 investigators and elsewhere indicate several promising avenues for regulation of photosynthate partitioning:

Sucrose biosynthesis and metabolism are key components of long-distance transport of photosynthates in vascular plants, and the extent of sucrose formation, metabolism, and sites of its conversion can markedly affect exporting and importing organs at many levels. Sucrose is typically synthesized in leaves via SPS (sucrose-P synthase) and cleaved at sites of import by either SuSy (sucrose synthase, a reversible reaction) or one of the invertases (in cell walls or vacuoles). Products of the invertase and SuSy paths differ (hexoses vs. fructose + UDPG), and so too does their potential to generate sugar signals. The Koch lab (FL-AES) has shown that these signals can repress genes for photosynthesis, and also affect invertase and SuSy genes themselves. In addition, sugar signals sensed through hexokinase can potentially be amplified by repeated cycles of sucrose cleavage and re-synthesis. Invertase action and the resulting signals are typically associated with growth, expansion and cell division, whereas SuSy is more often linked with biosynthesis of cell walls and storage materials. The Koch group (FL-AES) has shown that single and double maize mutants for the two major sucrose synthases (Sus1 and Sh1) showed previously unrecognizable vegetative phenotypes. Both mutants, which were typically characterized by their reduced kernel weight, also showed a significant reduction in plant height without affecting leaf number or node number. Cellulose content of cell walls was also reduced.

Florida-AES and Illinois-AES have addressed sucrose partitioning between invertase and sucrose synthase paths of metabolism using experimental systems ranging from Arabidopsis to Agrobacterium tumors and maize. Work with Arabidopsis led to identification of a wall-associated kinase (WAK1) that regulates one vacuolar invertase and root growth, but none of the other 7 invertases or 2 sucrose synthases. Studies with Agrobacterium tumors and developing maize ovaries both showed a developmental progression of sucrose partitioning to first, vacuolar invertases, then, cell wall invertases, and finally, sucrose synthases during maturation and storage stages. Similar patterns also appeared in a number of other systems. In contrast, partitioning to sucrose synthase was maximal under low oxygen conditions in the maize endosperm, and in the peripheral cytoplasm of phloem sieve tubes where oxygen availability and mitochondrial function are also considered low. More recent work initiated an exploration of sucrose partitioning to UDPG and other nucleotide-sugar precursors for cell wall biosynthesis, which could be important for biomass productivity. A strength of maize as a research tool (in addition to its size) lies in its transposon mutability, and FL-AES has developed a Mu-mutagenic population (UniformMu) with special features optimized for identification and characterization of single-gene knockout mutants. The Huber grouo (IL-AES/ARS) has characterized the third major sucrose synthase, Sus3. Unique features of this enzyme are that it is found in the lead mid-vein and is strictly cytoplasmic in subcellular location unlike Sus1 and Sh1 which are partially membrane bound. Binding of Sus1 to membranes was stimulated by sugars suggesting that it may play a role in sugar-sensing in diverting sucrose towards cellulose synthesis.

The Huber (NC-AES) and Chollet (NE-AES) groups have found that SuSy proteins from both maize and soybean nodules are phosphorylated at multiple sites; CDPKs and/or SnRKs have been implicated in these phosphorylation events. A collaborative study by Koch (FL-AES) and Huber (IL-AES) on determining the exact roles of Susy enzymes in sucrose metabolism modified at specific phosphorylation sites are being carried out in transgenic maize.

Starch synthesis occurs in chloroplasts of leaves and amyloplasts of non-green starch storage tissues (e.g., seeds and tubers). The extent of starch formation and use can be linked to both export and import of photosynthates by different plant parts. Starch is typically a composite of both amylose (unbranched ±-l,4 glucan) and amylopectin (±-l,4 and ±-1,6 branched glucan). Its synthesis requires three enzymes: AGPase (ADP-glucose pyrophosphorylase), starch synthase, and BE (branching enzyme). AGPase is an important regulatory enzyme and in many tissues it is activated by 3-P-glycerate and inhibited by inorganic phosphate. The Okita (WA-AES) and Preiss (MI-AES) groups have analyzed the roles of the different subunit types that makeup this enzyme with regard to catalytic and regulatory properties, and the Okita and Edwards groups (WA-AES) have found that starch content can be elevated in developing rice seeds by altering the properties of AGPase in transgenic plants. Kochs group (FL-AES) has shown that low-oxygen environment inside maize kernels favors C-partitioning to starch vs other metabolic fates. Endogenous hypoxia also favors C-deposition in the endosperm rather than the developing embryo. Oxygen levels inside developing endosperm were below detection by oxygen microprobes beginning with the earliest analyses at 10 days after pollination. Experimental increases and decreases in external oxygen levels were combined with additional data on internal oxygen levels, ATP concentrations, metabolite profiling, and 14C-sucrose labeling studies. Data indicated that C-partitioning to endosperm starch and its precursors was unaffected by further oxygen depletion, but that elevated oxygen levels shifted partitioning to developing embryos and their lipids. Guiltinen (PA-AES) lab has identified single and double mutants for the starch branching enzymes Sbe1 and Sbe2a which have help defined their roles in starch structure.

Partitioning of photosynthates to sugar alcohols is a central feature of several plant species that shunt recently fixed C into acyclic polyols such as mannitol and sorbitol, or cyclitols such as ononitol. In addition to their transport and storage roles, the Bohnert (IL-AES) and Loescher (MI-AES) groups have shown that these assimilates function as osmoprotectants for plants experiencing salt and water stress. Stress-susceptible model or crop plants have been transformed with genes involved in the synthesis of these protectants by the Bohnert and Loescher labs, and while stress resistance is often observed, more detailed studies of the mechanisms are required. Polyol transport and compartmentation require specific transporters. Loescher (MI-AES) has isolated and characterized a number of sorbitol transporters from apple and cherry and shown that they are important for fruit quality. In collaborative studies with Loescher (MI-AES), Edwards (WA-AES) have shown that transgenic Arabidopsis plants expressing mannitol 6-reductase are more tolerant to salinity through analysis of photosynthesis and various growth parameters. Gillaspy (VA-AES) has been studying the gene families of enzymes responsible for the biosynthesis and metabolism of myo-inositol, a sugar alcohol linked to osmoprotective functions, signal transductions and ascorbic acid biosynthesis. Ongoing studies have shown that the second enzyme of the pathway, myo-inositol monophosphatase hydrolyzes L-galactose 1-phosphate to form the precursor for ascorbic acid synthesis via the GDP-mannose pathway. Preliminary results from Kochs group (FL-AES), together with evidence for hypoxia in maize kernels, indicate a possible role for sucrose partitioning to sorbitol in the maize kernel. Among other possible advantages, this sugar alcohol can provide a valuable shuttle for transfer of excess reducing power from the hypoxic endosperm to the better-oxygenated embryo. Metabolite analyses, labeling studies, and other preliminary data are consistent with a biosynthetic role for sorbitol dehydrogenase (SDH) in the maize endosperm where it is abundantly expressed and enzymatically active. This possibility will be further tested in proposed studies.

Objective 4: Developmental and Environmental Limitations to Photosynthesis

The aim of this research is to analyze the limitations and environmental factors that influence photosynthetic productivity at the whole plant and canopy levels. Particular emphasis will be placed on abiotic stresses (temperature, water, and salinity), nitrogen use, and global atmospheric change. This work will integrate understanding developed here and under objectives 1 through 3 to optimize photosynthetic production and yield under current and future environmental conditions. The collaborating units include KS-AES, IL-AES, NV-AES, MN-AES, MI-AES, PA-AES, VA-AES (7 institutions, 9 research labs).

Factors that enhance or limit agricultural productivity do so by impacting photosynthesis. Gains in yield of the major crops over the past half-century have been predominantly through improved harvest index (HI). HI has approached the maximum achievable for many crops, and future gains will depend on increasing total production. This, in turn, will depend on improving the efficiency of crop canopy light interception and utilization. It will therefore be important to maximize these efficiencies under optimal and stress conditions, consonant with global atmospheric change.

NC-1142 collaborators have been active in a number of investigations that have explored developmental and environmental limitations to photosynthesis. These include nitrogen use, water use, stress (temperature, salt, drought, CO2), and developmental factors associated with canopy architecture and leaf ontogeny. Nitrogen acquisition and use is key to increasing actual efficiencies of light interception and use; improvements in nitrogen use efficiency (NUE) will also reduce the input of fertilizer needed for high yields. The Below group (IL-AES) has been studying the Illinois Protein Strains of maize -a germplasm with a wide range of NUEs - and found that little genetic variation exits for NUE in corn, probably because current hybrids have been selected under high N. The Below group has utilized kernel development in maize to understand how N supply and developing sink tissues influence the establishment and maintenance of the photosynthetic apparatus.

Another focus on plant development and photosynthesis, taken by the Guiltinan group (PA-AES) has identified maize starch-branching enzyme mutants which will impact future improvement of crop productivity. Results from these mutants suggest that alteration of starch branching patterns alters the normal source sink relationships within leaves.

Stresses represent major limitations to photosynthesis and yield. The Jones group (MN-AES) has developed biomarkers for heat stress, that may also be used to detect changes in osmotic and salt stress. To understand mechanisms of plant responses to both rising atmospheric carbon dioxide concentration and tropospheric ozone, with particular reference to photosynthesis and relating changes at the molecular and biochemical level to observations of whole systems in the field, IL-AES (the Long group) has continued their long-term studies on global change. A major focus over the past two years has been examining the interactive effects of predicted 2050 levels of ozone and CO2 on soybean and Arabidopsis under fully-open air field conditions using the SoyFACE facility. Analysis of larger scale FACE experiments has revealed that while rising CO2 increases photosynthesis and yield, the increases even in a nitrogen-fixing crop are smaller than anticipated, while the simultaneous increase in ozone results in a substantial yield loss.

NC-1142 collaborators have also addressed the impacts of salt stress and drought on plant productivity. The Bohnert (IL-AES) and Cushman (NV-AES) groups have pioneered the use of microarrays, gene mining, and mutant analyses to investigate the mechanisms of drought and salt stress and the role of osmoprotectants in these processes. For example, collaborative studies have elucidated the gene expression changes that occur during salt and drought tolerance in whole plants. The Guiltinan group (PA_AES) has pursued similar work in the agronomically important species, Theobroma cacao . The Gillaspy group (VA-AES) has examined the molecular signaling pathways that operate during drought stress, and identified inositol signaling genes that can impact ABA sensitivity of plants. The Aiken group (KS-AES) has examined similar physiological events, but in the context of crops grown in the field. Studies establishing baseline data for relative growth rates and responses of crops to drought have been performed and will establish the basis for rapid screening and identification of advanced crop lines in the germplasm development process.

Objectives

1. Examine the dynamic regulation of radiant energy capture and utilization in photosynthesis, and to study the architecture, function and biogenesis of the photosynthetic apparatus.
   
2. Determine and modify the biochemical and regulatory factors that impact the photosynthetic capture and photorespiratory release of CO2.
   
3. Understand the mechanisms that regulate photosynthate partitioning into paths for biosynthesis and use of sucrose, starch, and sugar alcohols.
   
4. Analyze the limitations and environmental factors that influence photosynthetic productivity at the whole plant and canopy levels.
   

Methods

Objective 1: Plastid Function and Intracellular Communication Suppressors of IMMUTANS and VAR2 will be generated using map-based cloning methods, and the suppressor mutants will be characterized at the molecular, biochemical and physiological levels (Aluru et al., 2006). These studies will identify components of the regulatory network that governs chloroplast biogenesis. The role of HXK-like proteins in glucose signaling will be studied by Moore (SC-AES) using functional genomic approaches of targeted mutants and transgenics, coupled with biochemical approaches to examine interacting proteins with a presumed scaffold for a possible signal complex. These studies will clarify mechanisms of glucose signal perception and signal propagation. The Moore lab will also characterize glucose-repressed seedlings using biochemical and molecular approaches in order to identify how glucose modulates cellular mitochondrial levels. The Jang lab (OH-AES) will focus on elucidating the role of bZIP transcription factors in the transcriptional cascade activated by glucose signaling. Downstream target genes of the bZIP transcription factors and any conserved cis-acting elements will be identified by chromatin immunoprecipitation coupled GeneChip analysis. The Jang and Moore labs will continue to exchange seed stocks for functional analyses, with particular emphasis on identifying read-outs for glucose signaling and the coupling of upstream and downstream signaling components. Identifying horizontal gene transfer (HGT) from V. litorea to E. chlorotica will be one focus of the Rumpho lab (ME-AES). To do this, key photosynthetic V. litorea nuclear gene sequences will be used to design primers for PCR and probes for S- and N-blotting, as well as fluorescent in situ hybridization (FISH) to sea slug DNA. The Rumpho lab will also collaborate with the Rodermel lab (IA-AES) to carry out a comparative chloroplast proteome analysis on algal chloroplasts and sea slugs chloroplasts (covering the 10 month symbiotic association). Since there are no algal nuclei in the sea slug, this analysis will allow us to study the influence of nuclear regulation on both the stability of chloroplast proteins and de novo synthesis of chloroplast protein complexes. Objective 2. Photosynthetic Capture and Photorespiratory Release of CO2 The Spreitzer group (NE-AES) will place all large-subunit phylogenetic residues together in groups based on their van der Waals interactions in the high-resolution Chlamydomonas Rubisco structure. These 30 mutant enzymes will be created by directed mutagenesis and chloroplast transformation. Coupled with new findings on the role of the small subunit in Rubisco catalysis, targeted regions will be subjected to random mutagenesis and DNA shuffling, and methods will be developed for the genetic selection of an improved Rubisco enzyme. The potential for producing an improved Rubisco in crop plants may be realized by the further development of Rubisco transformation by the Portis group (IL-ARS). The role of posttranslational hydroxylation and methylation of the Rubisco large subunit will be investigated by isolating Rubisco from Chlamydomonas and crop plants grown under different environmental conditions, and analyzing the enzymes with respect to mass spectrometry and enzyme kinetics (NE-AES, KY-AES). Continued collaboration (NE-AES, IL-ARS, AZ-ARS) on the structural interactions between Rubisco and Rubisco activase will focus on creating directed substitutions in the Chlamydomonas large subunit (in vivo) and Arabidopsis small subunit (in vitro) that will allow chemical crosslinking studies. The Houtz group will continue to explore and document the diversity of alternative protein substrates for Rubisco LSMT and the associated effects on enzyme activity by utilizing tobacco Rubisco LSMT knock-out plants. The essentiality and importance of DEF in the co-translational processing of chloroplast-proteins will be characterized and further studied using transgenic tobacco plants over-expressing DEF. Recent studies demonstrate that at least half of total chloroplast-localized DEF is bound to thylakoid ribosomes. Finally the design and synthesis of specific DEF inhibitors for the creation of ternary complexes with DEF and subsequent active-site characterization using X-ray crystallography will be pursued. The Portis and Savucci groups will collaborate in investigating the biochemical basis for Rubisco deactivation in response to moderate heat stress and to determine the potential of altering activase to reduce the inhibition of photosynthesis by moderate heat stress and to thereby increase plant productivity. In the coming year the Cushman group will focus on two major topic areas: 1) Integrative functional genomics of CAM: We plan to develop integrated functional genomic tools to identify and functionally test key regulatory components that control the unique metabolic demands of CAM. We propose to 1) provide a comprehensive description of mRNA expression profiles over a diel time course in plants performing C3 photosynthesis and following induction of CAM using large-scale EST sequencing and oligonucleotide microarray-based expression monitoring, 2) to identify changes in protein expression profiles in leaves and specifically identify low abundance proteins that play critical roles in CAM using state-of-the-art proteomics methodologies, 3) to conduct comparative metabolite profiling in ice plant leaves to identify differences between C3 and CAM modes of photosynthesis, and 4) to identify the vacuolar malate channel responsible for nocturnal vacuolar acidification and investigate the mechanisms by which the influx and efflux of C4 acids through acidic vacuoles are regulated. 2) CAM evolution: We are in the process of investigating the molecular genetic changes that have occurred during CAM evolution by mapping the occurrence of both weak and strong CAM species by 1) conducting simultaneous molecular phylogenetic analysis using DNA sequence data, foliar carbon isotopic composition, and nocturnal acid accumulation measurements; 2) developing novel molecular markers to trace the evolutionary progression of CAM and correlate these data with traditional, quantitative diagnostic indicators of CAM and existing molecular phylogenies; 3) surveying expression changes that are associated with the C3 to CAM evolutionary progression using oligonucleotide microarrays in closely related species that perform C3 photosynthesis, weak CAM and strong CAM; and 4) elucidating changes in cis-acting elements that are diagnostic of CAM evolution. Objective 3: Mechanisms Regulating Photosynthate Partitioning The steps involved in C-flow into and out of sucrose, starch, and sugar alcohols as well as lipids represent key points of control for photosynthate partitioning at the metabolic and whole plant levels. The proposed research will define mechanisms of regulation for each of these processes, and how they can be manipulated to increase productivity and yield. Regulation of sucrose biosynthesis and metabolism will be approached in several ways. Regulation can be modulated in both exporting and importing tissues: for example, at points of sucrose formation via SPS (sucrose-P synthase) or cleavage by SuSy (sucrose synthase) or invertases (vacuolar and cell wall forms). Invertase regulation and its role in development will be further examined using transgenic maize in collaborative research by the Koch (FL-AES) and Huber (IL-AES) groups. In these experiments, several types of transgenic lines will be generated: a) invertase promoter-GUS constructs for analysis of transcriptional control (FL-AES, IL-AES); and b) lines that over-express soluble invertase in specific tissues to examine the developmental role of invertase (FL-AES). A final regulatory aspect of sucrose metabolism that will be investigated is SuSy phosphorylation in coordinated research by the Koch (FL-AES) and Huber (IL-AES) groups. Stable and transient expression approaches will be used to test functionality, localization, and substrate characteristics of non-phosphorylatable SuSy enzymes. Both maize and soybean will be utilized (vegetative and nodule tissues), together with antibodies to specific sites of SuSy phosphorylation (developed by the NC- and NB-AESs). Of particular interest will be the identification of the effector kinase (a CDPK or SnRK). Fl-AES, IL-AES and IA-AES will build on the usefulness of maize and Arabidopsis plants that show altered expression of invertases and sucrose synthases. Arabidopsis work from FL-AES and Iowa-AES will appraise the physical and molecular phenotypes of single- and double- knockout mutants for the vacuolar invertases. Data thus far show differences in root elongation and expression of other genes for sucrose metabolism. Maize work from FL-AES and IL-AES will characterize transgenic maize lines that overexpress or silence sucrose synthases. Effects of VP1-mediated ABA signaling will also be tested on sucrose partitioning during early reproductive growth of the vp1 ABA-insensitive maize mutant (initial results indicate altered invertase expression). In addition, FL-AES will screen the UniformMu maize DNA grids for mutants in key genes that could affect sucrose partitioning. Regulation of starch biosynthesis will include a major focus on AGPase due to its central role in starch formation. Studies on the role of the two subunit types that make up this oligomeric enzyme will be continued by the Okita lab (WA-AES). Specifically, the role of the non-catalytic AGPase large subunit in effecting the catalytic activity of the AGPase small subunit will be investigated. The large subunit binds the substrate ATP and studies will be designed to determine whether this subunit also binds the second substrate glucose 1-phosphate and effectors 3-PGA and Pi. Edwards (WA-AES) and Okita will continue their studies on the effects of starch metabolism on plant growth and development in Arabidopsis and rice. Transgenic Arabidopsis plants over-expressing AGPase accumulate higher levels of leaf starch and have higher growth rates than wildtype plants. Studies will be conducted to determine whether starch metabolism influences photosynthetic capacity in these transgenic plants. Transgenic rice plants over-expressing AGPase produce larger seeds reflecting higher starch levels. No further increase in seed weight is seen in plants fixing CO2 at higher rates indicating a limitation in carbon flux from sucrose into starch. WA-AES will carryout metabolomic studies to determine what biochemical reactions are limiting starch synthesis. Koch (FL-AES) will test mechanisms by which the endogenous hypoxia in developing kernels (Rolletschek et al., 2005) could be exerting its influence on C-partitioning. This group will focus on avenues for dissipating excess reducing power (NADH) and on metabolic shuttles that could transfer this to the better-oxygenated embryo. Mutants in starch biosynthesis will be examined in conjunction with studies proposed above for sucrose partitioning and sugar-alcohol metabolism. Guiltinan (PA-AES) will focus our research on the characterization of the role of SBE during leaf development and the regulation of SBEs during development and throughout the diurnal cycle. In particular this lab will be interested in the transition from sink to source in developing leaves and how this is regulated. Potential protein to protein interactions and protein phosphorylation of the SBEs will be investigated. The fine structure and digestibility of mutant starches will be studied with a view towards possible applications of these genotypes in development of resistant starches for foods. Regulation of lipid biosynthesis will focus on efforts to maximize carbon flux into storage lipids. Benning (MI-AES), a new investigator of the project, has identified a WRI1 transcription factor from Arabidopsis which controls primary metabolism in seeds and is required for seed oil biosynthesis (Cernac et al., 2006). Proposed studies will include the (i) development of an in vitro system to study the interaction of the WRI1 factor with its target promoters and (ii) attempt to re-direct carbon flux into oils in non-seed tissues by ectopic expression of WRI1. The role of polyol sugars in imparting protection from drought or salt stress will be studied in coordinated efforts by the Bohnert (IL-AES) and Loescher (MI-AES) groups using stable transgenic model and crop plants. Photosynthetic characteristics of transformed tobacco and Arabidopsis plants producing mannitol, sorbitol, or ononitol will be assessed after salt stress. The importance of having cytosolic or chloroplastic polyols will be examined further via the expression of specific transporters (MI-AES). These experiments will define the protective characteristics of each polyol and the preferred compartment for its synthesis and accumulation, thereby providing insights into the use of polyols for engineering stress tolerance. Recent progress in regeneration and transformation of celery (a mannitol syntheziser) and cherry (a sorbitol synthesizer) by the Loescher group (MI-AES) will allow the study of genetically modified polyol biosynthesis under stress and during fruit development. FL-AES will test the hypothesis that sucrose partitioning to sorbitol in maize endosperm may have a previously unrecognized function for shuttling excess reducing power (NADH) from the hypoxic endosperm to the better-oxygenated embryo. Preliminary evidence indicates that sorbitol moves from endosperm to embryo throughout most of kernel development (apparent by 10 days after pollination), and that endosperm conditions favor its biosynthesis by the reversible sorbitol dehydrogenase (SDH) enzyme. Data show SDH activity in the endosperm, SDH gene expression there, and an apparent single gene encoding this activity in maize and rice. For planned studies, a putative Mu-knockout of the SDH gene in maize will be isolated, and a detailed analysis of this SDH mutant in conjunction with other genetic, metabolic, and labeling studies will be conducted to determine sorbitol function in maize kernels. The polyol myo-inositol has been suggested to be a precursor for ascorbic acid. To obtain conclusive evidence for this relationship, Gillapsy (VA-AES) will determine if the myo-inositol monophosphatase (IMP) enzyme is bifunctional in hydrolyzing both inositol-1-P and L-galactose-1-P (a precursor for ascorbic acid) and by examining IMP loss of function mutant plants to determine how substrate and product levels are affected by a loss of individual isoforms. To determine how the MIPS genes are used in inositol metabolism, loss-of-function mutants for MIPS1, MIPS2 and MIPS3 genes will be identified and characterized and the impact on myo-inositol synthesis assessed. The metabolic and visible phenotypes of these mutants, as well as any impacts on ascorbic acid synthesis will be studied. Objective 4: Developmental and Environmental Limitations to Photosynthesis NC-1142 collaborators will focus on the impact of five major developmental and environmental limitations to photosynthesis: 1. Nitrogen use. Experiments will be performed to understand how the N supply regulates leaf N mobilization and to discover the metabolic pathways and genes that respond to N, and thus which are associated with N use efficiency in maize (IL-AES). The IL-AES will utilize a well-established in vitro culture system to manipulate the amount, time, and form of N available to developing maize kernels. The developmental and physiological responses of key transport amino acids will be examined along with changes in gene expression via microarrays and directed quantitative RT-PCR assays of candidate genes. Developmental investigations by PA-AES will complement the above work. Further examination of leaf and pollen phenotypes of starch-branching enzyme mutants will examine whether protein phosphorylation is a control mechanism for starch-branching enzymes in maize leaves. 2. Temperature stress. The KS-AES will investigate effects of chilling on seedling emergence and subsequent growth in sorghum. Comparative analysis of breeding lines exhibiting differential chilling responses will establish thermal response benchmarks for seedling emergence, photosynthesis, assimilation and relative growth rate, prior to V6 development. 3. CO2 and ozone stress. Tropospheric ozone is thought to account for $6 billion of crop losses in the US, and its continued rate of increase might eliminate any increase in production due to rising CO2. The Free-Air Concentration Enrichment experiment (SoyFACE) has been examining ozone (1.5x ambient) to its elevated CO2 treatment (1.5x ambient). This first field system worldwide that simultaneously treats crops in the open air with both ozone and CO2 elevated to predicted future levels. To analyze larger scale FACE experiments to test whether the prediction that the detrimental effects of global change in the corn belt may be offset by increases due to rising CO2 is over-optimistic. IL-AES is developing improved modeling temperature functions for predicting photosynthesis that will provide a basis for the development of better mechanistic models of crop responses to global climate change. The Bohnert group, also part of IL-AES will evaluate Arabidopsis and Thellungiella halophila in SoyFACE conditions to determine the transcriptome differences in a drought tolerant species. 4. Salt and drought stress response. The role of signal transduction in stress sensing and tolerance responses will be studied by IL-AES, VA-AES and NV-AES. IL-AES is taking a forward genetics approach to identify novel signal transduction components in drought signaling. Examination of signaling processes that are controlled by phosphoinositide kinases will be explored, as these proteins appear to occupy an important switch between the stress response and continued growth. VA-AES will examine phosphoinositide metabolite patterns in plants exposed to drought and ABA to determine how genes that regulate phosphoinositides impact stress signaling. Phosphoinositides are implicated in intracellular calcium release, which activates the calcium-dependent protein kinases (CDPKs) studied by the NV-AES. The Cushman group will characterize CDPK interacting proteins and their role in drought sensing. A focus on functional analysis of drought tolerance genes from resurrection plant (S. lepidophylla) will provide novel targets for future genetic engineering efforts to address drought stress. Other NC-1142 participants (Moore, SC-AES, Jang, OH-AES, and Koch, FL-AES) are also addressing signal transduction pathways that exhibit cross-talk with drought and salt signaling pathways. Thus we expect for these investigators studies described in elsewhere to facilitate progress in understanding how these stress pathways limit photosynthetic function. 5. Water-use efficiency. IL-AES will examine the importance of the aquaporin genes in Arabidopsis using an RNAi approach to determine the mechanism of how aquaporins facilitate water transport in plants. The Aiken group (KS-AES) will identify mechanisms accounting for differential transpiration efficiency (leaf and whole-plant basis) among USDA sorghum accessions. Comparative analysis will focus on evidence for bundle sheath leakage, AvCi relationships, stomatal responses to VPD, canopy architecture and assimilate utilization processes.

Measurement of Progress and Results

Outputs

• Objective 1: <ul> <li>Genetic suppressor mutants for IMMUTANS will be identified, as well as other proteins that interact with IMMUTANS (IA-AES). <li>Glucose binding affinity of the hexokinase proteins and the organization of their putative signal complex will be determined (SC-AES). <li>Mechanisms for glucose control of plastid gene expression and mitochondrial abundance will be identified (OH-AES, SC-AES). <li>The bZIP transcription factor network will be characterized with regard to glucose response and targeted DNA binding elements (OH-AES). <li>The extent and significance of horizontal gene transfer to sea slug DNA will be determined using a candidate gene approach (ME-AES). <li>Chloroplast proteins will be identified which are uniquely stable and/or contributed during endosymbiosis by chloroplast DNA (void of algal nuclear control) or nuclear DNA of E. chlorotica (IA-AES, ME-AES). </ul>
• Objective 2: <ul> <li>All regions, relatively far from the Rubisco active site, that influence catalytic efficiency will be defined (NE-AES). <li>The role of Rubisco posttranslational modifications will be elucidated as well as other alternative substrates for Rubisco LSMT (KY-AES, NE-AES). <li>The highly desirable and economical use of plant peptide deformylase as an alternative to antibiotic selectable markers in plant transformation will be demonstrated. The potential for plant peptide deformylase as a target for a new class of broad-spectrum herbicides will be demonstrated. <li>Directed mutagenesis and chloroplast transformation in Chlamydomonas will be used to modify the Rubisco large subunit to allow the physical interactions between Rubisco and Rubisco activase to be defined (IL-ARS, AZ-ARS, NE-AES).<li>The biochemical factors contributing to Rubisco deactivation in response to moderate heat stress will be determined. <li>The effects of expressing altered forms of activase on the respone of photosynthesis to moderate heat stress will be determined. <li>A functional analysis of selected members of the CDPK and Di19 gene families as well as members of the hydrophilin (or late embryogenesis abundant or LEA) protein family from Arabidopsis and S. lepidophylla will be determined by loss-of-function and gain-of-function assays in Arabidopsis. <li>Protein-protein interaction studies will be expanded to include a large-scale, random all-against-all yeast two-hybrid screens and targeted screens for unknown gene products that are regulated by abiotic stress. <li>Protein-protein interaction studies using in vivo TAP tagging approaches to gather complementary interaction data sets for selected genes will be determined. </ul>
• Objective 3: <ul> <li>Transgenic maize with altered genes for invertases (FL-AES) and sucrose synthase will be generated (Fl-AES, NC-AES). <li>A method for absolute quantitative RT-PCR of all 8 Arabidopsis invertases. Transgenic Arabidopsis and rice plants with altered AGPase activity and starch storage capacity in leaves and seeds will be generated (WA-AES). <li>Novel maize genotypes containing altered starch structure and physical properties will be produced (PA-AES). <li>Transgenic maize plants with altered expresson patterns of various starch branching enzyme combinations will be produced (PA-AES). <li>Define the interaction of WRI1 transcription factor with its cognate promoter elements. Shift carbon metabolism towards oil biosynthesis by ectopic expression of WRI1 transcription factor. <li>The role of polyol transporters for sorbitol and mannitol will be defined under stress conditions and during fruit development (MI AES). <li>Loss of function mutants for various genes of the myo-inositol pathway will be generated and studied to determine the role of this polyol in plant growth and development and in ascorbic acid biosynthesis.</ul>
• Objective 4: <ul> <li>Genes or pathways will be discovered that are associated with or that potentially regulate NUE, storage product

Outcomes or Projected Impacts

• Objective 1: <ul> <li>Further insight into the function of IMMUTANS in photosynthesis, plant development and plant stress responses might lead to the design of strategies to manipulate the photosynthetic capacity and quality of important crop plants. <li>Understanding the control of mitochondrial biogenesis is a key step to understanding plant bioenergetics and possible exploitation of elevated CO2 growth conditions. <li>Elucidating glucose activated transcriptional cascades and the function of the glucose-responsive transcription factor that promotes floral transition could allow manipulation of key cellular and developmental processes. <li>Identifying expressed plastid proteins and acquired plastid functions during endosymbiosis between E. chlorotica and V. litorea will establish the framework for targeting nuclear processes in crop species that might enhance photosynthesis. </ul>
• Objective 2: <ul> <li>Genetic engineering of the Rubisco small subunit, post- and cotranslational modifications, and/or interactions with Rubisco activase might increase carboxylation efficiency and crop plant productivity (NE-AES, KY-AES, IL-ARS). <li>A deeper knowledge of regulatory phosphorylation and PEPC structure/function relationships in C4 and CAM plants might allow CO2 concentrating mechanisms to be genetically improved or transferred to C3 crop plants (NE-AES, NV-AES).</ul>
• Objective 3: <ul> <li>A new method for 3-anchored 454 expression profiling. <li>The substrate specificity of myo-inositol monophosphatase will be examined to determine whether it can produce a precursor for ascorbic acid. <li>A mutagenic maize population (UniformMu) optimized for identification and characterization of single-gene knockout mutants. <li>Modified genes for AGase with regulatory properties that facilitate greater starch production will be produced (WA-AES). <li>Identification of a gene for regulation of vacuolar invertase and root growth.</ul>
• Objective 4: <ul> <li>Genes or pathways will be discovered that are associated with or that potentially regulate NUE, storage product deposition, and salinity tolerance (IL-AES, NV-AES, VA-AES). <li>Genotypes, including growth forms, will be identified that have increased yields under atmospheric change (IL-AES, KS-AES). <li>Novel genetic determinants from resurrection plants will be identified. <li>Further characterization of the pathways associated with N stress and kernel growthpromises to reveal new strategies for improving nitrogen use efficiency in maize and other cereal crops via plant breeding and biotechnology approaches (IL-AES). </ul>

Milestones

(0): Establishment of protein interaction assays and their implementation in years 1 and 2 will be necessary to allow follow-up testing of early events that control plastid biogenesis (IA-AES). Recombinant HXK and HXK-like proteins will need to be made by year 2 to provide material for proteomic analyses and characterization of glucose binding affinities (SC-AES). Chromatin IP methodology will need to be refined by year 2 to identify partner-specific DNA targets and to test with key transgenics and mutants in years 3 and 4 (OH-AES). Algal probes for nuclear and chloroplast genes will be developed by year 2 for use in expression and FISH assays in years 3 and 4 (ME-AES). Plastid proteomic analysis procedures will need to be optimized by year 2 and applied in years 3 and 4 to study nuclear and chloroplast protein contributions to endosymbiosis (ME-AES).

(0): All phylogenetic regions of the Chlamydomonas Rubisco large subunit will be defined by year 2. These regions will then be subjected to random mutagenesis, coupled with genetic selection, as a means for improving the catalytic efficiency of Rubisco (NE-AES). The environmental dynamics of posttranslational hydroxylation and methylation of the Chlamydomonas Rubisco large subunit will be elucidated by year 3 (NE-AES, KY-AES). Enzymes with various states of modification will then be characterized with respect to kinetic properties. Directed-mutant substitutions will be created in the Chlamydomonas large subunit by year two, which will then allow crosslinking studies with Rubisco activase (AZ-ARS, IL-ARS, NE-AES). Knowledge of all biochemical pathways contributing to Rubisco deactivation will be required by year two to fully analyze the effects of altering activase on the effects of moderate heat stress on photosynthesis.

(0): New methodologies for both PCR- and 454-based maize mutant screens will be developed and implemented by year 2. One or more maize Mu-knockouts for SDH (sorbitol dehydrogenase) will be obtained by year 3. A series of DNA grids for targeted, specific-gene, knockout screens of over 17,000 UniformMu maize mutants will be developed by year 2.

(0): Germplasm will be developed with increased photosynthetic potential under elevated CO2 and ozone for use in breeding programs (IL-AES). Corn germplasm will be developed for increased NUE that might save growers money spent on N fertilizer and decrease N runoff (IL-AES).

Projected Participation

View Appendix E: Participation

Outreach Plan

The major advances and discoveries of the proposed research will be published in scientific journals and meeting proceedings. Peer-reviewed publication is the best practical method for evaluating the quality and impact of the research results. NC-142 investigators have been successful in this endeavor, as illustrated by the large number of articles published by NC-1142 members in high impact, high quality journals (see APPENDIX 1 and 2). Research results will also be presented as platform lectures and poster presentations at local, regional, national, and international scientific meetings. As another outreach effort, to foster exposure to plants and plant-based research, an inquiry-based plant science outreach module will be developed and delivered to elementary school students. NC-1142 members will participate as scientist partners in Virginia Techs Partnership for Research and Education in Plants (PREP) program by donating mutant Arabidopsis seed and engaging high school students and teachers in research projects.

Organization/Governance

The recommended Standard Governance for multistate research activities include the election of a Chair, a Chair-elect, and a Secretary. All officers are to be elected for at least two-year terms to provide continuity. Administrative guidance will be provided by an assigned Administrative Advisor and a CSREES Representative.

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Attachments

Land Grant Participating States/Institutions

CA, FL, IA, IL, KS, KY, LA, MD, ME, MI, MS, NE, NV, OH, PA, WA, WI

Non Land Grant Participating States/Institutions

Michigan State University, USDA-ARS/Arizona