NC_old1200: Regulation of Photosynthetic Processes
(Multistate Research Project)
NC_old1200: Regulation of Photosynthetic Processes
Duration: 10/01/2012 to 09/30/2017
Statement of Issues and Justification
Necessity of Photosynthesis Research
Photosynthesis is essential to life on earth as it converts sunlight into biochemical energy used by the vast majority of all life forms. It is the primary process for generating plant biomass. As a result of photosynthesis, carbon dioxide is converted into reduced forms of carbon (e.g., sugars, lipids, amino acids and other cell building blocks) and oxygen is produced through photosynthetic splitting of water. As such, plant and algal photosynthesis affects global geochemical processes, in particular carbon cycling (42), and is an important factor to be considered in global climate change models. Aside from these fundamental aspects of photosynthesis, agricultural production of food, fibers, natural chemicals, and biofuel feed stocks is directly affected by limitations of photosynthesis (130,147). It is widely recognized that some of the greatest challenges humankind is currently facing -feeding an exponentially growing global population, supplying sufficient energy to sustain this global population, averting negative environmental impacts due to human activities- can be addressed using photosynthetic organisms (i.e. agricultural crops) (39). Therefore, the need for state-of-the-art photosynthesis research to improve the efficiency of the process in traditional crops or for the development of novel crops has never been more urgent.
Collaborators participating in this regional project will place considerable focus on understanding and improving the response of photosynthesis to developmental and environmental factors that limit productivity. The research spans all levels of organization from the molecular and cellular through the leaf, whole plant and canopy levels. Particular emphasis will be placed on abiotic stresses (i.e., heat, cold, drought and salinity), nitrogen and water use efficiency, and the signaling pathways that initiate the plant response. 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 practices, particularly the application of fertilizer (50). For well-fertilized crops, HI has approached the maximum achievable for many crops, and future gains will depend on increasing total production (127). Greater productivity, which is necessary to meet the food, fuel and fiber needs of a growing world population (103), requires improving the basic efficiency of the photosynthetic process for light energy capture and the utilization of this energy for the synthesis of organic molecules (127). For maximum benefit, the efficiency of the process must be increased under both optimal conditions and when conditions are sub-optimal, consonant with the changes in temperatures, CO2 and precipitation anticipated from climate change.
Importance and Consequences
It has become increasingly clear that global climatic trends are negatively impacting the yields of our major crops used for the production of food, feed, and biofuels (83). Sustaining the productivity of traditional crops will require improvements in the efficiency of photosynthesis that go beyond traditional breeding and selection (94). Similarly, the development of novel feedstocks for biofuel/chemicals production in a way that is sustainable, that is not in competition with food, and that reduces greenhouse gas production requires novel approaches and non-traditional crops including possibly algae (118,134). Only a concerted and vertically integrated effort encompassing all aspects of photosynthesis by plant scientists will ensure that appropriate solutions will 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 crops 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 and fuels in a rapidly changing climate.
Genomic resources and second generation sequencing technologies have advanced to the point that a wealth of information can be generated for any species of plant or alga (e.g. date and oil palm) (3,16), in a relatively short time span and at reasonable costs. Thus, the raw material for genetic engineering of novel crops or algal strains is readily available. Large-scale phenomics focused on chloroplast proteins in model plants such as Arabidopsis has become possible (85). Moreover, using comparative genomics, reconstruction of metabolism from gene expression and genomic data has become readily feasible for any organism. One example is the recent metabolic reconstruction of the alga Chlamydomonas reinhardtii (22). In addition, our knowledge and analysis of primary metabolism of plants and metabolic networks has advanced to levels (78) that can enable the rational design of novel crops, which will meet our needs. Despite this progress, the task of genetically transforming crop plants and analyzing the consequences (phenotypes) as the result of genetic engineering is still tedious and time consuming, and synthetic biology efforts with plants involving stacking or replacing multiple genes are lagging behind those for bacterial systems (98). 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 the basic discovery process and to devise strategies for the improvement of photosynthesis in crops, facile model organisms such as Arabidopsis (121) or micro alga such as Chlamydomonas (99) will have to be employed to quickly identify the most promising directions for crop improvement. Such an integrated approach requires multifaceted expertise and, thus, will benefit from synergy derived from a multi-investigator effort.
Multi State Effort
Providing a conceptual framework through the current project, this North Central Regional Group of scientists has already successfully worked on diverse aspects of photosynthesis bringing together a complementary set of expertise. While global issues as laid out above are addressed, practical solutions to these problems often have local solutions that take 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 North Central Region and other participating states. Participating partners are listed below for each focus area.
Efforts by the group are organized into four themes (Objectives). While the details and outcomes will be discussed in detail in the main body of the proposal, likely impacts falling under these themes can be briefly summarized as follows:
1. Plastid Function and Intracellular Communication. The chloroplast is the organelle in plant and algal cells that harbors the photosynthetic apparatus. It is also the location for a large number of enzymes turning this organelle into a biochemical factory. Biogenesis of the chloroplast requires the cooperation with other cell organelles. The import of nuclear encoded proteins or membrane lipids assembled at the ER provide two examples (14,75). As primary photosynthate and many other metabolites (e.g. fatty acids) are only synthesized in the plastid, they have to be exported to be available to other cell compartments. Furthermore, 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. Specific studies will focus on the photosynthetic electron transport chain, lipid and protein transfer phenomena involving plastids, as well as signaling pathways affecting chloroplast biogenesis and function. Likely impacts are an increased understanding of protein and lipid transfer processes involved in chloroplast biogenesis, as well as an improved understanding of the signals and regulatory processes that adjust photosynthetic electron transport or are required for chloroplast biogenesis and homeostasis. (IA-AES, KS-AE, MI-AES, NE-AES, WSU-AES).
2. Photosynthetic Capture and Photorespiratory Release of CO2. Photosynthetic carbon fixation and photorespiratory release of CO2 have long been recognized as limitations to crop productivity (49,95,100,119). Considerable focus will be placed on engineering improvements in ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and the heat-sensitive activase that regulates its function. Microbial CO2 concentrating mechanisms will be investigated for potential improvements of algae and crop plants. Emphasis will also be placed on the mechanisms that control carbon flux through primary and secondary metabolic pathways. Likely impacts will be insights into the regulation of carbon fixation and the generation of modified enzymes that improve primary and secondary carbon metabolism in plants. (AZ-ARS, IA-AES, IL-ARS, LA-ES, NE-AES, NV-AES, WA-AES).
3. Mechanisms Regulating Photosynthate Partitioning. Manipulation of carbon partitioning and understanding its regulation is central to the design of new biofuel crops that, for example, divert carbon from carbohydrates into triacylglycerols (38). Transport phenomena at cellular, tissue, and whole plant scales will be considered, sugar sensing mechanisms will be explored, as well as partitioning between storage carbohydrates, lipids and stress-protective compounds. Likely impacts will be defining the mechanisms that regulate photosynthate partitioning into the biosynthetic pathways for sucrose, starch, sugar alcohols, and lipids. Strategies will be developed to alter carbon partitioning by engineering. (FL-ARS, Il-ARS, MI-AES, MN-AES, MS-AES, VA-AES, WA-AES).
4. Developmental and Environmental Limitations to Photosynthesis. Factors such as leaf anatomy (126) or environmental stress-factors such as high light (90), excess salinity or high temperature (112) greatly affect photosynthesis. Particular emphasis will be placed on abiotic stresses (temperature, water, and salinity), nitrogen use, and global atmospheric change. Likely impacts will be defining the environmental factors that influence photosynthetic productivity at the whole plant and canopy levels. (IL-AES, KS-AES, MI-AES, MN-AES, NV-AES, AZ-ARS).
Related, Current and Previous Work
Comprehensive CRIS database searches were performed by individual participants using specific and more general search terms such as photosynthesis alone (812 hits alone) and in combination with other terms such as lipids (21 hits), RuBisCo (60 hits), carbon fixation (40 hits), etc. were conducted as relevant to the topic. Aside from projects associated with NC1168 members, no apparent overlap with projects by others was detected. It was apparent that no other regional project offers the same focus or breath of multistate participation in covering this topic.
1. Plastid Function and Intracellular Communication.
Chloroplast biogenesis and function of the photosynthetic membrane is a prerequisite for plant photosynthesis. Collaborators under Objective 1 focus on different aspects of this process. The aim of the Rodermel lab (IA-AES) is to characterize proteins that regulate chloroplast biogenesis. The focus will be on PTOX and VAR2, both of which are thylakoid membrane proteins that play a central role in photoprotection of the photosynthetic apparatus (6,142). These proteins were identified ~10 years ago by cloning the genes responsible for two Arabidopsis variegations (immutans and var2); gene isolation and characterization were carried out under the previous NC-1168 Project (23,136). PTOX (the IMMUTANS gene product) is a quinol terminal oxidase that participates in the control of the redox poise of the plastoquinone pool; it acts as a safety valve to shunt excess electrons from the PQ pool to oxygen, forming water (5,43,87). VAR2, on the other hand, is an FtsH-like metalloprotease that is involved in D1 turnover during the PSII repair cycle (23). It also mediates a number of membrane modeling events that occur during thylakoid membrane biogenesis.
The Stone lab (NE-AES) is interested in studying chloroplast biogenesis by determining the function of the DJ-1 protein in chloroplast development. Arabidopsis contains three close homologs of DJ-1 originally identified in animals, where it is causally involved in different diseases such as Parkinsons (15). Homozygous disruption of the AtDJ1C gene results in non-viable, albino seedlings that can be rescued by expression of wild-type or epitope-tagged AtDJ1C (79). The plastids from the dj1c mutant plants lack thylakoid membranes and grana stacks, indicating that AtDJ1C is required for proper chloroplast development. The essential role for AtDJ1C in chloroplast maturation expands the known functional diversity of the DJ-1 superfamily and provides the first evidence of a role for specialized DJ-1-like proteins in eukaryotic development.
Photosynthetic membranes contain predominantly galactolipids (34) and the Benning lab (MI-AES) focuses on the assembly, biosynthesis and turnover of thylakoid lipids and the regulation of these processes. Two pathways are involved the biosynthesis of lipids of the photosynthetic membrane: 1. Assembly of glycerolipids from de novo synthesized fatty acids in the chloroplast; and 2. Assembly of lipid precursors at the endoplasmic reticulum (ER) followed by import into plastids (14). To address the question of the mechanism for the transport of lipids from the ER to the inner chloroplast envelope membrane, mutants of Arabidopsis partially disrupted in the import of lipid precursors from the ER were isolated (140). Severe mutant alleles cause embryo lethality indicating the essential function of the respective proteins (139). All mutants isolated to date carry mutations in one of four proteins, TGD1, 2, 3, and 4. Their name, TriGalactosylDiacylglycerol, is derived from the accumulation of a diagnostic oligogalactolipid in the mutants due to the activation of a processive galactosyltransferase (SFR2) , known to be involved in lipid remodeling required for freezing tolerance in plants (88). The TGD1, 2, and 3 proteins are components of an ABC transporter complex associated with the inner envelope membrane that likely bridges the interenvelope membrane space (9,84,140). TGD4 is a predicted beta barrel protein that is part of a distinct protein complex associated with the outer envelope membrane (138). It is hypothesized that these TGD proteins form two interacting complexes giving rise to a conduit for PtdOH transfer from the ER to the inner envelope membrane of plastids.
Work in the Kramer lab is aimed at understanding how the photosynthetic machinery is integrated into plants and algae to provide precisely the right amount of energy, in precisely the correct forms, without self-destruction. To accomplish this, the Kramer lab has developed or harnessed a set of powerful in vivo spectroscopic tools to probe key components of photosynthetic electron and proton transfer, ATP synthesis, photoprotection, photoinhibition etc. (11). Using these tools, the importance of regulation of the ATP synthase and cyclic electron flow in balancing the energy budget, efficiency and protection of photosynthesis has been established (66,70,81,82,104,125,144). The Kramer lab will harness these tools for high-throughput photosynthetic phenomics (high-throughput phenotyping) of both algae and plants, to study photosynthesis at a systems level, and to use this technology to determine how the photosynthesis network is regulated in response to fluctuating environmental conditions. This approach will allow the assessment of downstream regulation at the biochemical and biophysical levels and to determine the roles of previously unknown genes, proteins, and processes in setting the regulatory strategy of energy transduction. Importantly, this novel phenomics platform will be available to members of the NC1168, for example, for the analysis of lipid mutants
2. Photosynthetic Capture and Photorespiratory Release of CO2
The common goal is to improve the rate of CO2 fixation by focusing on the primary events of CO2 acquisition (49,89,95,119). Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of photosynthesis by fixing CO2 from the atmosphere. The Spreitzer (NE-AES) group has defined structure-function relationships of Rubisco by employing genetic approaches in the green alga Chlamydomonas reinhardtii, which is the only eukaryote that allows genetic engineering of both nuclear and chloroplast-encoded subunits of Rubisco. Guided by phylogenetics and high-resolution X-ray crystal structures, regions have been identified that might serve as targets for improving the Rubisco enzyme (64,106). More recently, catalytically-improved plant/algal hybrid enzymes have been produced (45), indicating that it might be possible to express plant Rubisco in Chlamydomonas for pursuing new strategies aimed at improving the crop-plant enzyme.
Rubisco is maintained in an active state by another chloroplast protein, Rubisco activase. Rubisco activity might be increased by altering the interaction of Rubisco with activase or improving activase's thermal stability (94,119). The Salvucci (AZ-ARS) group is elucidating the structure-function relationships of activase and its interactions with Rubisco. Past work (AZ-ARS, NE-AES) identified a substrate recognition domain in activase that directly interacts with a region in Rubisco (74,93). In a collaborative effort between Salvucci (AZ-ARS) and Wachter's group at Arizona State University, the X-ray crystal structure of this lever-arm domain has been solved recently (53). The Spreitzer (NE-AES) and Salvucci (AZ-ARS) groups are investigating the interaction between activase and Rubisco, particularly the involvement of the Rubisco small subunits, the Rubisco central solvent channel, and the Rubisco large-subunit carboxyl terminus. To understand activase's extreme thermolability, Salvucci (AZ-ARS) has found that activity loss upon moderate heating is accompanied by aggregation and is affected by Mg2+ and nucleotide levels (13).
Chlamydomonas has an inducible CO2 concentrating mechanism (CCM) that concentrates CO2 in the vicinity of Rubisco, thereby decreasing photorespiration and increasing net CO2 fixation. Several project participants are investigating the CCM to determine whether or not it can be engineered to increase algal growth for the production of biofuels, or whether or not a CCM might be introduced into crop plants (IA-AES, LA-AES, NE-AES). The Spalding group (IA-AES) has been investigating the LCIB protein (in complex with LCIC) that is distributed in the chloroplast stroma at low CO2, but concentrates around the pyrenoid at very low CO2 concentration. Suppressors of the lcib-mutant have been recovered and two are allelic with thylakoid CAH3 carbonic anhydrase (36,37). Collaborative efforts by Spalding and Weeks (NE-AES) have identified an ABC-type transporter, HLA3, that is located in the plasma membrane and is involved in HCO3- transport (35). This work also implicated the plastid envelope protein, LCIA, in HCO3- transport. Transcriptome sequencing has identified additional candidates that might function in the CCM, especially additional Ci transporters (IA-AES, NE-AES). As a means for engineering CCM genes, Spalding and Weeks have collaborated on developing transcription activator-like effector nucleases (TALENs) for genome editing in Chlamydomonas (77). The Moroney (LA-AES) group has used insertional mutagenesis to study the CCM. Nine carbonic anhydrase genes and genes that might encode HCO3- transporters have been identified. Moroney and Ynalvez (89) have proposed a model for CCM function. The Moroney lab has also studied genes encoding carbonic anhydrase isoforms, three of which have been found to be important for the acquisition of CO2.
In C4 and Crassulacean acid metabolism (CAM) plants, phosphoenolpyruvate carboxylase (PEPC) catalyzes the initial fixation of CO2, thereby concentrating CO2 near Rubisco. The Cushman (NV-AES) group has investigated the molecular basis of CAM evolution and the roles of storage carbohydrates and circadian clock control of CAM. CAM evolution progresses from C3 photosynthesis to weak and then to strong CAM. Strong CAM is correlated with reciprocal fluxes in acidity and storage carbohydrates, tissue succulence, isogene recruitment, and leaf-specific and circadian clock controlled mRNA expression (115-117). A colorimetric assay was developed to measure leaf pH and screen a fast neutron-mutagenized population of common ice plant (Mesembryanthemum crystallinum) to isolate CAM-deficient mutants (27). One mutant showed greater H2O2 content and Cu/Zn-superoxide dismutase mRNA expression, indicating that CAM might contribute to alleviation of oxidative stress (124). Feeding of sugars restored nocturnal accumulation of organic acids, indicating that CAM possesses some flexibility for utilizing different carbohydrate sources (27). Gene expression profiling showed changes associated with induction of CAM by salinity stress (28). Many genes undergo shifts in their circadian-clock output phase during the stressed-induced shift from C3 photosynthesis to CAM providing new CAM-specific markers.
An unusual mechanism of carbon acquisition is found in some C4 species in the family Chenopodiaceae. These species exist as single cells rather than the dual-cell Kranz anatomy required in C4 crop plants. Edwards and Okita (WA-AES) have been defining all aspects of this novel C4 system (e.g., (92)), which may provide ideas for introducing a C4 CO2 concentrating system into C3 plants.
3. Mechanisms Regulating Photosynthate Partitioning.
For the regulation of photosynthate partitioning three levels of control are relevant. First, metabolic controls have been explored by IL-ARS, MI-AES and WA-AES focusing on specific enzymes. Of particular interest are sucrose and amino acids because the biosynthetic pathways for both end products involve at least one enzyme that is controlled by protein phosphorylation, sucrose phosphate synthase (SPS) (123) and nitrate reductase (NR) (55), respectively. IL-ARS has determined that both enzymes have a conserved methionine (Met) residue within the phosphorylation motif that directs the requisite protein kinase to the regulatory phosphorylation site on the enzyme. Phosphorylation of these enzymes is thought to be sensitive to reactive oxygen (ROS) signals (e.g., H2O2) via oxidation of the essential methionine residue. If so, this could be a general link between conditions that increase ROS species, such as abiotic stress, and photosynthate partitioning.
Starch synthesis is also an important target for manipulating source-sink relationships as a means to increase the genetic yield potential of crop plants (54,129) and plant productivity in general. Past work has established that two regulatory enzymes, ADP glucose pyrophosphorylase (AGPase) and phosphorylase I, control different phases of starch biosynthesis. AGPase catalyzes the first committed step in the starch biosynthetic (maturation) pathway and is subjected to allosteric regulation and redox control. Over the past 18 years WA-AES has made considerable strides in understanding the roles of the large and small subunits that comprise the heterotetrameric subunit enzyme structure using a biochemical-genetic approach (12,51,56-60,67). Expression of a highly catalytically active, but regulatory-insensitive bacterial AGPase during rice seed development increased seed weights of 8-15% (105). Similar increases in seed weight were evident when an up-regulated form of the potato AGPase large subunit was expressed in developing rice seeds (73). Mass spectrometry analysis of the major metabolites in transgenic rice seeds indicated that expression of the bacterial AGPase increases the flux of carbon into starch by elevating the steady-state concentration of metabolites present at near equilibrium.
Work by MI-AES on leaf starch degradation pathways (24,111,120,131-133) showed that beta-maltose is the primary molecule for export of carbon from chloroplasts at night. Export of maltose in preference to triose phosphates or glucose lowers the ATP required for starch to sucrose conversion at night from four to three (131). The starch degradation pathway provided important insight into sugar signaling mechanisms because leaf starch metabolism is a major source of glucose for the sugar-sensing hexokinase reaction (111). It also resulted in an understanding of the potential roles of phosphorolytic (to supply chloroplast metabolism) and amylolytic (for export from the chloroplast) leaf starch breakdown (132).
A second level of control of carbon partitioning is sugar-mediated signal transduction. Research from FL-AES focused on differential sugar-responsiveness of genes for sucrose metabolism, and C/N balance, to produce a global framework for selective advantages of feast-and-famine genes in multicellular organisms (68,69,141). In support of the overall goals, work by FL-AES also helped establish a national resource for corn (Zea mays L.) knock-out mutants that compares with that for Arabidopsis (MaizeGDB.org (86,110))
Work from several laboratories including OH-AES and VA-AES have identified components of the sugar-mediated signal transduction pathway as targets for increasing plant yield (61,62,113). OH-AES has focused on the dissection of hexokinase-dependent and independent sugar signaling mechanisms (62,137), signal crosstalk between sugar and hormones (97,146), and more recently the transcription factor networks involved in sugar signal transduction (63,96). A family of sugar responsive Tandem Zinc Finger (TZF) genes are regulated by hexokinase-dependent sugar signaling mechanisms (25), and might play key roles in integrating sugar/hormone signal transduction and RNA regulation in gene expression (80). The sucrose non-fermenting related kinase-1 (SnRK1) (122) gene encodes a highly conserved energy and stress sensor that VA-AES and other groups have identified as a key regulator of sugar-mediated signaling in plants (7,8,10).
A third level of control over carbon partitioning involves the carbon sink compounds called polyols. MI-AES has shown that plants with the capacity to synthesize various acyclic polyols have substantial tolerance for salt and drought stress (41,44). Transgenic plants with elevated acyclic polyol biosynthesis contain substantial improvements in salt tolerance (20,114). Conversely, plants with reduced concentrations of different polyols can be hyper-sensitive to stress and grow poorly (33,128). Thus the study of polyol synthesis and the impact of various polyols on plant growth, development and stress responses might yield valuable insights for altering carbon partitioning.
4. Developmental and Environmental Limitations to Photosynthesis
NC-1168 collaborators have been active in a number of investigations that have explored developmental and environmental limitations to photosynthesis. These include nitrogen and water use efficiency and stress physiology (e.g., heat, salt, drought, cold), as well as the underlying mechanisms that signal plants to respond to stress. Nitrogen acquisition and use are key to increasing the efficiency of light interception. Improvements in nitrogen use efficiency (NUE) will lower production costs by reducing the input of fertilizer needed for high yields, which in turn will decrease the amount of pollution associated with fertilizer run-off. Research by the Below group (IL-AES) is focused on agricultural intensification using advanced maize and soybean genetics to increase yields with reduced fertilizer inputs, lowering the amount of N required per unit of carbon fixed. This group has investigated the impact of N and maize genetics on the partitioning of photosynthates into yield (109). Their work suggests that kernel composition is source limited in maize. Determining the nitrogen mass balance for a tile-drained agricultural watershed in east-central Illinois showed that soil N depletion is likely to occur in years with above-average precipitation or extremely wet spring periods (47).
Breeding and selection for improved plant performance under different stresses can be accelerated by the development of high-throughput methods for phenotyping plant responses (102). The Aiken and Prasad groups (KS-AES) have used theoretical consideration to simulate stomatal behavior based on radiation utilization (1,2) and have shown that canopy temperature and chlorophyll fluorescence show potential as drought tolerance screening tools. The research includes field evaluation of a procedure to quantify the stay-green trait (18), a drought resistance feature, in sorghum. Salvucci's group (AZ-ARS) has used a tractor-based platform for monitoring canopy temperature, normalized difference vegetation index (NDVI), and plant height to examine the response of 25 cotton cultivars to the combined effects of heat and drought stress.
Tolerance/adaption to both cold and heat are addressed in the NC1168 project. Cushman's group (NV-AES) is working with Camelina sativa, a potential biofuel crop, to develop germplasm with increased cold tolerance. Research on high temperature stress is a major focus of the NC1168 with the Sharkey (MI-AES), Salvucci (AZ-ARS), Cushman (NV-AES), Loescher (MI-AES), Aiken (KS-AES), Prasad (KS-AES) and Harper (NV-AES) groups all participating in the effort. Sharkeys group has used both lab and field studies to uncover the effects of heat on electron transport (108,135). This group has shown that under heat stress 1) photosystem I becomes more reduced and the stroma becomes more oxidized (107); 2) the proton motive force (pmf) decreases; and 3) the proportion of the pmf that is accounted for by the pH component decline (143,145). Salvuccis group (AZ-ARS) is focusing on the acute thermolability of Rubisco activase (13), which reduces the activation state of Rubisco under heat stress (71,72). This group has developed an assay for measuring activase activity in leaf extracts and used this assay to characterize the temperature response of activase from several plant species, including C. sativa (19). The groups of Aiken and Prasad (KS-AES) are investigating the effects of high temperature stress on photosynthesis and fruit-set (30,31). They have found that the inhibitory effects might be mediated through ethylene response through the production of reactive oxygen species and subsequent membrane damage (29,32). Loeschers group (MI-AES) has investigated the effects of heat stress on photosynthesis in grapevines. Their research showed that the effects could be attributed to inhibition of electron transport activities and a decrease in the activation state of Rubisco.
Stomatal behavior is central to both, drought stress and water use efficiency, and is being investigated actively. Efforts include phenotyping studies by Aiken (KS-AES) and Salvucci (AZ-ARS), as well as efforts by Prasad (KS-AES) to elucidate the basis of the slow wilting trait in sorghum (91), and to examine the response to water vapor pressure deficit (48). Cushmans group (NV-AES) is focused on improving drought stress in C. sativa using a variety of approaches to increase its cultivation range.
The Cushman group (NV-AES) is also investigating, Dunaliella salina Teodoresco, a unicellular, halophytic green alga that is among the most industrially important microalgae. This algae produces massive amounts of b-carotene and is a potential feedstock for biofuel production (101). To understand the molecular basis of its salinity tolerance and its potential as a biofuel feedstock, this group obtained ESTs for 2,831 clones representing 1,401 unique transcripts from a complementary DNA (cDNA) library (4) to capture mRNA expression under high and low light and dark conditions, NaCl, anaerobic, and nutrient deprivation stress.
The response of plants to abiotic stress is communicated at the cellular and molecular levels through a network of signaling pathways, often involving protein kinases. The Harper group (NV-AES), in collaboration with Cushman (NV-AES), has obtained evidence for in vivo interactions between a 14-3-3 and 121 different clients, raising the total number of putative 14-3-3 clients in plants to over 300 proteins (21,26). Many of these interactions are involved in regulating subcellular organization, signaling and metabolic regulation and most require phosphorylation of a binding site on the client. Many of these phospho-interactions are likely mediated by Calcium-dependent Protein Kinases (CPKs) (17). The Li group (MS-AES) has identified a Vicia faba abscisic acid-activated protein kinase (AAPK) and they have shown it to be a positive regulator of abscisic acid (ABA)-induced stomatal closure (76). In a search of the targets of this kinase, they have identified several phosphoproteins regulated by ABA and drought (52,65). The identity of these targets suggests that ABA-activated protein kinases play an important role in the regulation of chlorophyll degradation and carbohydrate metabolism.
Determine factors that regulate the biogenesis and maintenance of chloroplasts and the assembly and function of the photosynthetic membrane.
Determine and modify the biochemical and regulatory factors that impact the photosynthetic capture and photorespiratory release of CO2.
Determine the mechanisms regulating photosynthate partitioning and to carbon partitioning by engineering.
Analyze the limitations to photosynthetic productivity caused by environmental factors.
MethodsObjective 1. Plastid Function and Intracellular Communication. The Rodermel lab (IA-AES) is interested in defining the regulatory mechanisms underlying PTOX and VAR2 function using immutans and var2 as tools. The principal method involves the generation and characterization of second-site genetic suppressors of these mutants. It is presumed that such suppressors define activities that bypass the need for PTOX and VAR2 because they are able to give rise to all-green, photosynthetically-competent plants. To date, over 100 var2 and five immutans suppressor lines have been isolated, and a dozen have been characterized at the molecular level. Many of these suppressors are components of the chloroplast translation apparatus (for var2 suppressors), or novel thylakoid membrane proteins (for var2 and immutans suppressors). Using molecular genetics approaches, the Stone lab (NE-AES) will focus on the analysis of knockout mutants for the A. thaliana DJ-1 homologs. At least one gene is essential; two AtDJ1C gene T-DNA insertion mutant alleles confer an albino, seedling-lethal phenotype, the wild-type protein is targeted to chloroplasts, and mutant albino plant tissues have severely disrupted chloroplast ultrastructure indicating that the protein functions in chloroplast biogenesis. Similar approaches will be used to analyze the functions of the AtDJ1C homologs. In addition, X-ray crystallographic techniques will be employed to determine the structures of recombinant proteins produced in E. coli. The Benning lab (MSU-AES) will explore mutants deficient in lipid trafficking, lipid remodeling and lipid assembly in Arabidopsis and Chlamydomonas. Lipid import from the ER to the chloroplast will be studied by developing a chloroplast-based lipid import system. In vitro reconstitution of lipid trafficking complexes and lipid remodeling enzymes will be pursued to gain a mechanistic understanding of the TGD proteins, SFR2 and related proteins involved in lipid assembly, lipid remodeling and lipid trafficking in chloroplasts. New mutant screens will be developed to identify additional factors required for the assembly of lipids of the photosynthetic membranes or lipid droplet formation in algae. Lipid mutants produced in the Benning lab will be investigated for possible photosynthesis defects and activity of photosynthetic electron transport chain components to determine the role of specific lipids in photosynthetic electron transport. This will be done in collaboration with T. Sharkey and D. Kramer (MSU-AES). The Kramer lab (MSU-AES) will apply their newly developed spectroscopic tools for high-throughput photosynthetic phenomics (high-throughput phenotyping) to both algae and plants, to study photosynthesis at a systems level. This technology will be applied to determine how the photosynthetic network is regulated in response to fluctuating environmental conditions. This approach will allow the assessment of downstream regulation at the biochemical and biophysical levels and the determination of the roles of previously unknown genes, proteins, and processes in setting the regulatory strategy of energy transduction. The Kramer lab will take advantage of the integrated nature of NC1168 to establish new collaborations with other NC1168 members that apply the emerging technology to the overall question of what controls the balance between photosynthetic productivity and photo-damage in a changing environment. The aim is to understand the importance of these properties for photosynthetic performance under fluctuating environmental conditions, asking specific questions, such as: What are the energy costs and benefits to the algal carbon concentrating mechanism in Chlamydomonas? Is lipid composition important for acclimation of photosynthetic machinery to fluctuating light and temperatures? What are the relative roles of regulation of the chloroplast CFO-CF1-ATP synthase and cyclic electron transfer to control and balancing of photosynthesis in different Arabidopsis ecotypes? Objective 2. Photosynthetic Capture and Photorespiratory Release of CO2 The Spreitzer group (NE-AES) will express functional crop-plant Rubisco in Chlamydomonas to serve as a model for genetic engineering. This work will be based on the use of hybrid-enzyme mutants that express plant small subunits (45). Coupled with new findings on the role of the small subunit in Rubisco catalysis (46), 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. Selection will be based, in part, on the use of CCM mutants in collaboration with IA-AES and LA-AES. Collaboration (AZ-ARS, NE-AES) on the structural interactions between Rubisco and Rubisco activase will continue. The Salvucci group (AZ-ARS) will continue to elucidate the structure of Rubisco activase, particularly as it relates to its mechanism of action. Collaboration (NE-AES, AZ-ARS) on the structural interactions between Rubisco and Rubisco activase will focus on creating directed substitutions in the Chlamydomonas large subunit carboxyl terminus (in vivo) that will allow chemical crosslinking and functional studies. The effect of redox control of activase on photosynthetic performance will be determined by adding redox-regulated elements to beta forms of activase. The Spalding group will use deep sequencing of pooled progeny to identify mutated genes in suppressors of the lcib mutant air-dier phenotype. The Spalding group will also use site-directed mutagenesis to explore the structure-function of LCIB and the LCIB/LCIC complex, collaborating with Tom Smith at the Danforth Center, who will solve a crystal structure for LCIB. Spalding (IA-AES) and Weeks (NE-AES) will over-express the HLA3 Ci transport protein in Chlamydomonas wild type and CCM-deficient mutants, and in other model systems, to help understand the function of this transporter. Weeks and Spalding will also continue efforts to develop TAL nucleases for use in Chlamydomonas and plants as genome editing tools. They will continue their collaboration to identify additional functional components of the CCM based on transcriptome experiments, and Weeks will use bioinformatics and computational biology tools to investigate changes in gene expression controlled by microRNAs, long noncoding RNAs and cis-acting elements in the promoters of CCM-associated genes. RNA fractions will be subjected to RNA-Seq analyses using an Illumina DNA sequencing instrument. Moroney (LA-AES) will localize carbonic anhydrases and Ci transporters by using epitope tags and GFP chimeras, and will determine whether or not a protein is redistributed after a switch to low-CO2 growth conditions. RNAi or gene knock-out technology (such as the use of TALENs developed by IA-AES and NE-AES) will be used to define the role of specific proteins in the CCM. Moroney will also continue to investigate the role of carbonic anhydrases in plants by measuring growth, CO2 fixation, and stomatal conductance in Arabidopsis knock-out lines. The full spectrum of gene expression changes that are associated with the C3 to CAM evolutionary progression will be characterized by the Cushman group (NV-AES). Increased, leaf-specific, and circadian-clock controlled mRNA expression patterns will be assessed using real-time, qRT-PCR for a set of well studied CAM marker genes (e.g., PEPC) and correlated with traditional diagnostic indicators of CAM. High throughput Illumina sequencing (RNA-Seq) will also be used. Cis-acting elements acquired (or lost) during CAM evolution will be identified by bioinformatic pattern matching tools. Integrated transcriptomic, proteomic, and metabolomics expression pattern data sets will also be collected from wild-type and CAM-deficient mutant plants performing C3 photosynthesis and CAM over both diel and circadian (constant temperature and light) conditions in order to assess gene family members that are recruited specifically for the CAM pathway and to evaluate the effects of storage carbohydrates on circadian clock output phasing. Selected cis-elements will be identified bioinformatically and cognate transcription factors will be tested for functional roles in controlling circadian-clock controlled mRNA expression patterns using gain-of-function and loss-of-function promoter::luciferase transient reporter assays in attached leaves. C4 photosynthesis increases CO2 assimilation and represses photorespiration under CO2-limited conditions. A major question is how this is accomplished in single-cell C4 systems, including development of two types of chloroplasts, and two cytoplasmic domains, and how CO2 is concentrated to repress photorespiration. Studies will encompass the analysis of the mechanism of chloroplast differentiation, kinetic properties of Rubisco and features of Rubisco activase through collaborations between Edwards (WA-AES), Okita (WA-AES), Spreitzer (NE-AES), and Salvucci (AZ-AES). Current studies (WA-AES) on photosynthesis in rice, a C3 crop, has raised questions about the effectiveness of photosynthesis under CO2 limited conditions (i.e., the relationship between the CO2 compensation point, Rubisco specificity factor, and degree of refixation of photorespired CO2), which will be studied through collaborations between Edwards (WA-AES) and Spreitzer (NE-AES). Objective 3. Mechanisms Regulating Photosynthate Partitioning. The Huber group (Il-ARS) will determine whether or not phosphorylation of key enzymes, including nitrate reductase (NR) and sucrose-phosphate synthase (SPS) is regulated in vivo by oxidation of Met residues near regulatory phosphorylation sites. For NR, two Met residues will be targeted for manipulation: Met538 at the +4 position, and Met544 at the +10 position. It is speculated that the oxidation status of either or both of these residues will impact the ability of kinases to phosphorylate NR at the Ser534 regulatory site. Site-directed mutagenesis and transgenic plants will be used. These experiments should yield insights as to the role of NR in stress adaptation, and provide the first in vivo test of the methionine redox switch hypothesis. The Okita group (WA-AES) will examine the roles of AGPase subunits in enzyme catalysis and allosteric regulation. Substrate binding properties will be determined using Isothermal Titration Calorimetry (ITC). The catalytic and potential regulatory properties of PHO1 will be discerned by determining the crystal structure of rice PHO1 complex with a malto-oligosaccharide and the effector ADP-glucose. Site-directed mutagenesis and biochemical analysis will be performed to assess the roles of the amino acid residues in the ADP-glucose binding site, which will be identified from the X-ray crystal structures and/or labeling experiments. These results will indicate how ADP-glucose can serve as a mixed competitive inhibitor of PHO1 activity and influence its role in starch grain biosynthesis. A new factor involved in starch synthesis will also be examined, the ADP-glucose transporter BT1. Of the downstream processes that limit carbon flow in AGPase-expressing transgenic rice plants, transport of ADP-glucose from the cytoplasm to the amyloplast is the most likely suspect based on available evidence in the literature. The AGPase-transgenic rice plants will be re-transformed with the maize BT1 transporter under two strong seed-specific promoters and resulting plants analyzed for seed weight increases and metabolite levels. The Sharkey group (MI-AES) will focus on the roles of starch and sucrose on intracellular interactions required for optimal photosynthesis, using genetic mutants incapable of starch or sucrose synthesis. Regulation of photosynthesis in these plants by the interactions among phosphate, proton motive force, and end product synthesis will be determined. Measurements of rates of photosynthesis and starch and sucrose synthesis will be made under Rubisco-limited, RuBP-regeneration-limited, and triose-phosphate-use-limited conditions. In addition to steady-state measurements, measurements will be made in response to transients (for example a jump up or down in light or CO2) to determine how plant responses change during short-term adaptations to changes in conditions. The Koch and Loescher groups (FL-AES, MI-AES) will define biochemical effectors of photosynthate transfer along phloem and post-phloem paths by analyzing sugar transporters and other genes using a combination of phloem expression profiling and mutant analyses and methods modified from previous work (40). Genes with implicated roles will be prioritized for mutant analysis by a reverse-genetics approach using the UniformMu resource in maize. A forward-genetics approach will also be employed by identifying causal mutations underlying phenotypes that include atypical accumulations of photosynthates in leaves. Post-phloem transfer of photosynthates will be investigated by approaches employing maize mutants to alter transport processes. The first will be guided by a combination of microarray analyses and expression profiles of transfer cells, and the second will focus on photosynthate transport through the phloem-free endosperm of developing maize kernels. Regulation of metabolic partitioning between polysaccharides, sugars, and lipids will be investigated in maize kernels (major sinks for photosynthates). This work will not only enhance our understanding of how sugar-metabolism is regulated in endosperm, but also will provide new mutants and transgenic lines with economically valuable kernel properties. Links between sugar signaling and photosynthate partitioning will be examined in conjunction with each of the metabolic objectives above. In each instance, additional maize mutants will be examined from the UniformMu maize population for potential to perturb sugar signaling in this system. To determine the roles of specific factors in sugar signaling, the Jang group (OH-AES) will dissect the relationship between AtTZF1 and the Mitogen Activated Protein Kinase (MAPK) cascade, an evolutionarily conserved stress signal transduction pathway. A MPK has been identified as AtTZF1 interacting partner. Molecular, cellular, and biochemical analyses will be conducted to determine if AtTZF1 is phosphorylated in vivo by a MAPK cascade, and if so, how it affects AtTZF1s subcellular localization and gene expression. To determine if AtTZF1 is involved in RNA regulation and transcriptional regulation, 3UTR tethered function assay, transcriptional activation/repression assay, and Systematic Evolution of Ligands by EXponential enrichment (SELEX) will be conducted, respectively. To test if TZFs can be useful tools in crop improvements, an integrative approach will be used to functional characterize soybean TZFs in hormone-mediated growth and stress response using gain- and loss-of-function analyses. Global identification of soybean TZFs target genes by expression profiling will also be conducted. A second major target of sugar signaling studies by NC-1168 is the SnRK1 kinase. The Gillaspy group (VA-AES) will further characterize proteins that impact SnRK1 stability by binding and targeting SnRK1 for proteasomal turnover. Targets already identified will be placed in a protein interaction network by using co-immunoprecipitations followed by mass spectrometry and genetic analyses. Transgenic plants containing hyper-stable SnRK1 will be constructed and tested for metabolic, biomass and stress response alterations. Polyols and related osmoprotectants will be examined by both the Loescher group (MI-AES) and Gillaspy group (VA-AES). MI-AES will determine the effects of drought and salt stress stress on metabolism and partitioning of putative osmoprotectants (e.g., sorbitol, mannitol, and other cyclic and acyclic polyols) with respect to several components of photosynthesis (e.g., gas exchange, stomatal behavior, and electron transport). Transgenes involved in metabolism and transport of cyclic and acyclic polyols will be transferred to plants and the applicable effects of these genes for contributions to stress tolerance will be assessed. VA-AES will pursue similar work on the myo-inositol synthesis pathway to examine the relationship between general growth and myo-inositol levels. Defining the regulatory mechanisms, especially the responsiveness of key genes and enzymes in polyol metabolism to developmental, temporal, partitioning, and environmental signals, should provide critical insights into how these genes and polyols function in the complexities of stress tolerance, and how they might be manipulated to improve stress tolerance and plant productivity. Objective 4: Developmental and Environmental Limitations to Photosynthesis NC-1168 collaborators will focus on the impact of five major areas associated with environmental limitations to photosynthesis: 1. Nitrogen Use. The focus of the research is on agricultural intensification using advanced maize and soybean genetics to increase yields with reduced fertilizer inputs per unit of carbon fixed as crop biomass. Experiments will be performed with high populations and sustainable management practices (reduced tillage and stover management) to elucidate relationships between N supply and leaf N mobilization. By identifying maize metabolic pathways and genes responsive to N, fungicides, and ethylene blockers, N use efficiency can be enhanced through selective breeding efforts and management decisions (IL-AES). Photoperiod-sensitive maize will be evaluated for extended-season photosynthesis, and for production of sugars, biomass, and bioenergy under low N fertilizer conditions. 2. Temperature Stress. The MI-AES will investigate effects of temperature on pmf, proton conductance, stromal redox status, and PSII and PSI function, using mutants that vary in membrane properties. The AZ-ARS will pursue the goal of improving the thermostability of Rubisco activase in the oilseed crop, Camelina sativa, collaborating with the NV-AES on heat tolerance in this species and the NE-AES on Rubisco activase-Rubisco interactions. AZ-ARS will investigate the timing of photosynthetic inhibition by heat and drought stress in field grown cotton plants, using both low- and high-throughput methods. KS-AES will utilize physiological techniques such as canopy temperature measurements, gas exchange instrumentation, leaf fluorescence, pollen viability, seed-set percentage, and harvest index to quantify effects of heat stress, and develop remote sensing methods for assessing stress responses in the field. 3. Salinity Stress in Plants and Algae. NV-AES will create a transcriptome sequence database for all tissues from Camelina sativa plants growing under a combined stress regime including salinity. By transforming C. sativa with potential stress protection genes, the limits of abiotic stress tolerance of Camelina (e.g., drought, heat, cold, salinity) can be established. For the algal research, native isolates will be collected from local wastewater treatment facilities, refined to axenic cultures, and evaluated for general growth characteristics and lipid and starch content. The ability of these algal isolates to tolerate high concentrations of the liquid fraction of anaerobically digested sludge will be evaluated. 4. Drought and Water-Use Efficiency. KS-AES will conduct field studies to evaluate genotypes of sorghum, wheat and soybean for tolerance to drought or heat stress. To assess drought stress, canopy conductance will be inferred from digital images of vegetative indices and thermal irradiance. NV-AES will use transcriptomics and transformation to determine and manipulate drought tolerance in C. sativa. AZ-ARS will refine high-throughput methods for determining canopy temperature to uncover drought-tolerant cotton germ-plasm. 5. Signaling in Stress Sensing. The role of signal transduction in stress sensing and tolerance responses will be studied by NV-AES and MS-AES. The MS-AES will identify the phosphoproteins regulated by ABA-activated protein kinases using T-DNA insertion knockout mutants and RNAi knockdown lines and then verify them as substrates of the ABA-activated protein kinases. The NV-AES will identify phosphorylation events that are associated with abiotic stress responses by investigating the role of CPKs in the phospho-regulation of plant development and metabolism, particularly the involvement of phospho-regulation of 14-3-3 interactions. This project will involve collaborations with the Cushman and Huber labs, and complement lipid signaling work by the Gillaspy lab.
Measurement of Progress and Results
- 1. Genetic suppressors of immutans and variegated2 will be isolated in Arabidopsis and characterized (IA-AES).
- 2. DJ1-like proteins and their mutants will be characterized, interacting protein partners identified, and a crystal structure for one of the proteins determined (NE-AES).
- 3. SFR2 and mutant forms of Arabidopsis and different crop plants and will be isolated and biochemically characterized (MSU-AES).
- 4. Mutants deficient in ER-to-plastid lipid trafficking and proteins involved in the process will be identified and characterized (MSU-AES).
- 5. Arabidopsis and algal lipid mutants as well as mutants affected in different aspects of photosynthesis will be analyzed using a phenomics platform (MSU-AES)
- Strains of Chlamydomonas will be created that express crop-plant Rubisco (NE-AES) and enzyme engineering and genetic selection will recover improved Rubisco (AZ-ARS, IA-AES, LA-AES, NE-AES).
- The physical interactions between Rubisco and Rubisco activase will be determined using transgenic Chlamydomonas (AZ-ARS, NE-AES).
- The thermal stability of activase and its interaction with Rubisco will be improved (AZ-ARS, NE-AES).
- TAL nucleases will be developed for genome editing of algae and plants (IA-AES, NE-AES).
- The structures of LCIB and the LCIB/LCIC complex will be elucidated (IA-AES).
- The functional role of putative Ci transporter HLA3 in the CCM will be delineated (IA-AES, NE-AES).
- Additional genes required for function of the CCM will be identified (IA-AES, LA-AES, NE-AES).
- Arabidopsis carbonic anhydrase mutants will be recovered and characterized for defects in CO2 acquisition (LA-AES).
- Gene expression patterns and complex regulatory networks will be characterized that are associated with the evolution of CAM (NV-AES).
- Circadian clock regulatory outputs in wild type and CAM-deficient mutants will define the temporal orchestration of CAM (NV-AES).
- The mechanism of development of two types of chloroplasts in single-cell C4 species will be defined (WA-AES, AZ-ARS, NE-AES).
- The CO2 compensation point in rice will be defined based on Rubisco specificity factor and degree of refixation of photorespired CO2 (WA-AES, NE-AES).
- Transgenic plants expressing mutants of nitrate reductase with altered residues surrounding a regulatory phosphorylation will be generated (Il-ARS).
- Substrate binding properties of AGPase subunits and three-dimensional structure of plant Pho1 will be obtained (WA-AES).
- Transgenic rice plants expressing both AGPase and BT1 will be generated (WA-AES).
- Mechanisms regulating primary partitioning between starch and sucrose its relevance for plastid function will be uncovered (MSU-AES).
- Data of expression profiling and microarray analyses of phloem and transfer cells of maize and Arabidopsis will become available (FL-AES, MI-AES).
- New maize mutants over-expressing genes that enhance photosynthate partitioning to starch under high-temperature stress will become available (FL-AES).
- Data sets on TZF-specific DNA and RNA binding sites in both Arabidopsis and soybean will become available (OH-AES).
- Transgenic plants expressing hyper-stabilized SnRK1 energy sensor will be generated (VA-AES).
- Transgenic plants altered in myo-inositol synthesis will become available (VA-AES).
- Improvements in salt and drought tolerance in plants transgenic for acyclic polyol biosynthesis will be provided (VA-AES, MSU-AES).
- Phenotyping methods (including remote sensing) will become available for evaluating plant performance in response to abiotic stress and fertilizer inputs (KS-AES, AZ-AES, IL-AES).
- Genes or pathways associated with water- and nitrogen-use efficiency and tolerance to temperature extremes, drought, and salinity will be uncovered and manipulated in established crops, and new biofuel crops like Camelina, and in algae (IL-AES, NV-AES, MS-AES, KS-AES, MI-AES, AZ-AES).
- New protein targets that are phos
Outcomes or Projected Impacts
- Novel factors critical for chloroplast biogenesis and thylakoid membrane formation will be uncovered advancing our basic mechanistic understanding of chloroplast biogenesis (Il-AES, NE-AES, MSU-AES).
- It will become clear to what extent the lipid composition of the thylakoid membrane will impact productivity during acclimation of the photosynthetic machinery to light or temperature fluctuations (MSU-AES).
- The relative roles of regulation of the chloroplast CFO-CF1-ATP synthase and cyclic electron transfer to control and balancing of photosynthesis will be determined providing novel insights into the limitations of photosynthetic productivity (MSU-AES).
- Genetic engineering of Rubisco and/or Rubisco activase will improve the interaction between these proteins to increase carboxylation efficiency and crop-plant productivity (AZ-ARS, NE-AES).
- Engineering of the CCM in algae, or the transfer of a CCM into higher plants, will improve CO2 assimilation and increase productivity and its energy costs in Chlamydomonas will become apparent (IA-AES, LA-AES, NE-AES, MSU-AES).
- TAL nucleases will become viable tools for genome editing of algae and plants (IA-AES, NE-AES).
- Understanding the molecular genetic basis of evolution and the temporal regulatory requirements for CAM will establish a framework for strategies to improve water use efficiency in crop plants (NV-AES).
- Introducing single-cell C4 traits into major C3 crops is expected to increase productivity (AZ-AES, NE-AES, WA-AES).
- Understanding Rubisco properties and refixation of photorespired CO2 in rice, a C3 crop and major source of food on a global scale, is expected to lead to enhanced productivity (NE-AES, WA-AES).
- Mutants from this project will provide the first opportunity to assess the role of motif recognition elements in vivo in terms of phosphorylation and 14-3-3 binding. (Il-ARS).
- New insights to the role of nitrate reductase in stress adaptation and associated mechanisms will be obtained. (Il-ARS)
- The roles of the AGPase large and small subunits in catalysis and allosteric regulation will be determined. Insights on the regulation of Pho1 will be obtained. (WA-AES)
- Information generated by the study of transgenic rice plants with enhanced seed weights will aid efforts to increase cereal yields. (WA-AES)
- Novel strategies will be developed towards the engineering photosynthetic efficiency and partitioning of photosynthetic products at biochemical and whole-plant levels, as well as enhanced partitioning of photosynthetic products to harvested plant parts or products. (MSU-AES).
- Molecular manipulation of components involved in sugar signaling and polyol pathways is expected to aid in the design and production of crops with enhanced productivity and stress tolerance (OH-AES, VA-AES, MSU-AES).
- Salt and drought resistant crop cultivars will be developed that use water efficiently, and reduce their dependence on high quality irrigation water (VA-AES, MSU-AES).
- Novel phenotyping methods for plant performance will be demonstrated in the screening for stress tolerant germplasm and will lead to a refinement of our understanding of the impacts of various abiotic stresses under field conditions (KS-AES, AZ-AES, IL-AES).
- New stress tolerant crop and algal germplasm, including germplasm with higher nitrogen- and water-use efficiency, will be developed by manipulating the genes or pathways associated with environmental limitations to photosynthesis (IL-AES, NV-AES, MS-AES, KS-AES, MI-AES, AZ-AES).
- New strategies for developing stress tolerant germplasm will be tested that are based on altering stress-sensing pathways (MS-AES, NV-AES).
Milestones(2013): Crop-plant Rubisco will be expressed in Chlamydomonas (NE-AES) One lipid mutant of Arabidopsis will be analyzed for photosynthesis defects (MSU-AES). Construction of transformation vectors containing Nia2 directed mutants and transformation of the nia2 knockout and wild type plants with the Nitrate reductase genes will be completed (IL-ARS). Approximately 20,000 new UniformMu maize mutants (Mu insertions) will be mapped, taken to homozygosity, tested for association with a visible phenotype and made publicly available as seed lines each year through MaizeGDB.org this year and each subsequent year. (FL-AES).
(2014): SFR2 proteins from a freezing tolerant plant and non-freezing tolerant plant will be compared and biochemically characterized (MSU-AES). DJ1 interacting partners will be identified during the first two years (NE-AES) Crop-plant Rubisco expressed in Chlamydomonas will serve as a model for improving Rubisco productivity (NE-AES). Transgenic rice plants expressing both un-regulated AGPase and maize BT1 will be generated (WA-AES). Generation of plants that make both sucrose and starch, plants that cannot make sucrose during the day (starch only), and plants that cannot make or break down starch (sucrose only) will be completed (MSU-AES). Genome sequencing of Dunaliella salina will be completed (NV-AES). Phosphoproteins regulated by ABA-activated protein kinases and CDPK will be identified (MS-AES, NV-AES).
(2015): New components of lipid trafficking complexes in the chloroplast envelope membranes will be identified (MSU-AES). The mechanism for activation of Rubisco by activase will be elucidated. (AZ-ARS, NE-AES) Key functional components of the Chlamydomonas CCM will be delineated. (IA-AES, LA-AES, NE-AES) Single carbonic anhydrase knock-out Arabidopsis lines will have been recovered. Double knock-out lines will then be constructed for physiological experiments (LA-AES). Transcriptome-wide changes in mRNA expression patterns will allow a comprehensive assessment of the genetic changes required for CAM evolution (NV-AES). Highly diffractible crystals of Pho1 must be obtained to enable its structural determination (WA-AES). Identification, sequencing, and characterization of genes involved in sugar signaling and polyol metabolism and transport (MSU-AES, VA-AES) New germplasm, engineered with a more thermostable Rubisco activase, will be produced and evaluated (AZ-ARS). Generation of Camelina varieties with improved drought and heat tolerance will be completed (AZ-ARS, NV-AES).
(2016): Analysis of photosynthesis for three algal lipid mutants will be completed (MSU-AES). Acquisition of sequence information for different classes of regulatory RNAs and computational evaluation of the RNA populations will become available and used to discern their individual roles in CCM regulation (NE-AES). Chlamydomonas CCM genes will be targeted for TALEN manipulation (IA-AES, LA-AES, NE-AES). Transgenic nia2 knockout and wild type plants expressing NR genes will have been analyzed for plant growth with nitrate or ammonium as sole N-source (IL-ARS). Complete genome annotation of Dunaliella salina will lead to the development of customized algal feedstocks for either biodiesel or bioethanol product streams (NV-AES). Promising candidates of phosphoproteins regulated by ABA-activated protein kinases and CDPK will be tested in transgenic plants (MS-AES, NV-AES). Double carbonic anhydrase knock-out Arabidopsis lines will have been recovered and their phenotype analyzed in physiological experiments (LA-AES).
(2017): Approximately 10 suppressors of immutans or var2 will have been analyzed (IA-AES). All DJ1-like proteins of Arabidopsis will have been characterized (NE-AES). A thorough understanding of the effect of temperature on pmf, proton conductance, stromal redox status, and PSII and PSI function will have been obtained from analysis of Arabidopsis mut
Projected ParticipationView Appendix E: Participation
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-1168 investigators have been successful in this endeavor, as illustrated by the large number of articles published in high impact, high quality journals (see Appendix). 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-1168 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. In addition, NC-1168 members at MSU will host undergraduate students during an annual summer intern program in plant genomics (www.plantgenomics.msu.edu). Nebraska NC-1168 members are participants in two summer workshop programs, one in the Plant Sciences and another in Algal Biology and Biotechnology for high school teachers and students.
The Standard Governance for multistate research activities will be implemented which includes the election of a Chair (organizes current annual meeting), a Chair-elect (organizes next annual meeting), and a Secretary (organizes the subsequent annual meeting). Rotating through these functions, all officers are 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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2. Aiken, R. M. and N. L. Klocke. 2010. Inferring transpiration control from sap flow heat gauges and the Penman-Monteith equation. Proceed 5th Natl Decennial Irrig Conf, ASABE
3. Al-Dous, E. K., B. George, M. E. Al-Mahmoud, M. Y. Al-Jaber and others. 2011. De novo genome sequencing and comparative genomics of date palm (Phoenix dactylifera). Nat.Biotechnol. 29:521-527
4. Alkayal, F., R. L. Albion, R. L. Tillett, L. T. Hathwaik and others. 2010. Expressed sequence tag (EST) profiling in hyper saline shocked Dunaliella salina reveals high expression of protein synthetic apparatus components. Plant Sci. 179:437-449
5. Aluru, M. R., D. J. Stessman, M. H. Spalding, and S. R. Rodermel. 2007. Alterations in photosynthesis in Arabidopsis lacking IMMUTANS, a chloroplast terminal oxidase. Photosynth.Res. 91:11-23
6. Aluru, M. R., F. Yu, A. Fu, and S. Rodermel. 2006. Arabidopsis variegation mutants: new insights into chloroplast biogenesis. J.Exp.Bot 57:1871-1881
7. Ananieva, E. A. and G. E. Gillaspy. 2009. Switches in nutrient and inositol signaling. Plant Signal Behav 2009/10/02:304-306
8. Ananieva, E. A., G. E. Gillaspy, A. Ely, R. N. Burnette and others. 2008. Interaction of the WD40 domain of a myoinositol polyphosphate 5-phosphatase with SnRK1 links inositol, sugar, and stress signaling. 148:1868-1882
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11. Baker, N. R., J. Harbinson, and D. M. Kramer. 2007. Determining the limitations and regulation of photosynthetic energy transduction in leaves. Plant Cell Environ. 30:1107-1125
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14. Benning, C. 2009. Mechanisms of Lipid Transport Involved in Organelle Biogenesis in Plant Cells. Annu.Rev.Cell Dev.Biol. 25:71-91
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16. Bourgis, F., A. Kilaru, X. Cao, G. F. Ngando-Ebongue and others. 2011. Comparative transcriptome and metabolite analysis of oil palm and date palm mesocarp that differ dramatically in carbon partitioning. Proc.Natl.Acad.Sci.U.S.A 108:12527-12532
17. Boursiac, Y., S. M. Lee, S. Romanowsky, R. Blank and others. 2010. Disruption of the vacuolar calcium-ATPases in Arabidopsis results in the activation of a salicylic acid-dependent programmed cell death pathway. Plant Physiol 154:1158-1171
18. Burke, J. J. 2007. Evaluation of source leaf responses to water-deficit stresses in cotton using a novel stress bioassay. Plant Physiol 143:108-121
19. Carmo-Silva, A. E. and M. E. Salvucci. 2011. The activity of Rubisco's molecular chaperone, Rubisco activase, in leaf extracts. Photosynth.Res. 108:143-155
20. Chan, Z., R. Grumet, and W. Loescher. 2011. Global gene expression analysis of transgenic, mannitol-producing, and salt-tolerant Arabidopsis thaliana indicates widespread changes in abiotic and biotic stress-related genes. J Exp Bot 2011/08/09:
21. Chang, I. F., A. Curran, R. Woolsey, D. Quilici and others. 2009. Proteomic profiling of tandem affinity purified 14-3-3 protein complexes in Arabidopsis thaliana. Proteomics. 9:2967-2985
22. Chang, R. L., L. Ghamsari, A. Manichaikul, E. F. Hom and others. 2011. Metabolic network reconstruction of Chlamydomonas offers insight into light-driven algal metabolism. Mol.Syst.Biol. 7:518
23. Chen, M., Y. Choi, D. F. Voytas, and S. Rodermel. 2000. Mutations in the Arabidopsis VAR2 locus cause leaf variegation due to the loss of a chloroplast FtsH protease. Plant J. 22:303-313
24. Cheng, L., R. Zhou, E. J. Reidel, T. D. Sharkey and others. 2005. Antisense inhibition of sorbitol synthesis leads to up-regulation of starch synthesis without altering CO2 assimilation in apple leaves. Planta 220:767-776
25. Coello, P., S. J. Hey, and N. G. Halford. 2011. The sucrose non-fermenting-1-related (SnRK) family of protein kinases: potential for manipulation to improve stress tolerance and increase yield. J Exp Bot 2010/10/27:883-893
26. Curran, A., I. Chang, C. Chang, S. Garg and others. 2011. Calcium-dependent protein kinases from Arabidopsis show substrate specificity differences in an analysis of 103 substrates. Frontiers Plant Sci 2:
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|Name: _________________________||Period: ___________________|
This test consists of 5 short answer questions, 10 short essay questions, and 1 (of 3) essay topics.
Short Answer Questions
1. What is on the porch at the bungalow in Westmore Street where Marlowe goes looking for Talley?
2. What time is it when Marlowe looks at his watch in the jail cell?
3. What nationality is the boy who comes into the Peacock Lounge to sell papers?
4. Why does Cooney want to beat up Marlowe, after Dobbs tells him to stop?
5. Where is Derace Kingsley when Degarmo and Marlowe get to the cabin?
Short Essay Questions
1. After leaving the crime scene, what does Degarmo try to convince the other police officer?
2. After Patton shoots at Degarmo, what does Degarmo do?
3. How does Marlowe get out of the apartment at the Granada?
4. Who does Marlowe call to verify where Kingsley has gone, once he guesses where it is?
5. How does Degarmo react when Marlowe tells him about Mrs. Fallbrook?
6. How does Grayson describe Dr. Almore's practice?
7. Why is Marlowe surprised that Mrs. Kingsley would need money from her husband?
8. What reason does Marlowe give Mrs. Kingsley that she'll miss him?
9. How does Degarmo act toward the sentries at the dam on the way to the cabin?
10. What does Marlowe say about Muriel's explanation of why she went back to Lavery's the morning after the murder?
Write an essay for ONE of the following topics:
Essay Topic 1
Marlowe learns the truth through investigation and interviewing. Discuss investigation and interviewing in the novel.
1) What techniques does Marlowe use to investigate? How does he approach interviewing people?
2) How do Marlowe's interviewing techniques vary in different situations, with different people?
3) What information is Marlowe able to acquire by talking to people? What makes his interviews effective or ineffective?
Essay Topic 2
The Lady in the Lake is written in the first person narrative point of view, from the perspective of Marlowe. Discuss the novel's point of view.
1) Why does the author choose the first person narrative point of view? Why is the story told from Marlowe's perspective?
2) What does Marlowe reveal to the reader, as narrator, and when? What does Marlowe keep hidden from the reader, though he may know it himself?
3) How does the first person narration affect the tone of the novel? How is it appropriate to the genre?
Essay Topic 3
Throughout the novel, Phillip Marlowe encounters corruption, both moral and ethical. Discuss corruption in the novel.
1) How are the police in the novel corrupt, and how does Marlowe perceive that corruption?
2) What kinds of moral corruption does Marlowe face among the people he comes into contact with? How is that moral corruption related to the corruption of the police?
3) How does Marlowe deal with temptations to corruption, such as Kingsley offering him money to cover up a murder?
This section contains 808 words
(approx. 3 pages at 300 words per page)


Since the last few months, this thought has been the crux of my thinking. It has changed me. Or at least that is the idea. Not sure if this is who I want to be. Or do I? What about all those expectations I have? From people, from friends, from myself? Should I be bothered if I simply have no expectations from anyone? Have I become a lost soul? What if I lose everything I hold dear and near to me?
I know for a fact that I indeed have lost a few. Or is it the other way around? What if I get what I want right now, but after a few more months/years I realize I never wanted this? So many questions, such a predicament!
Then again, our dilemma is that we hate change and love it at the same time; what we really want is for things to remain the same but get better.
I read somewhere that the best things in life are unexpected – because there were no expectations.
I think I will stick to it for now.