Brief Summary of Minutes of Annual Meeting

The meeting was held at the Donald Danforth Plant Science Center in Saint Louis, MO. Ru Zhang (DDPSC) began the meeting with introductions and Christoph Benning discussed the report format and completion.

Doug Allen, MO-ARS with the USDA lab at the Danforth Center reported on ongoing investigations in algal metabolic flux. The lab continues to study C3/C4 central carbon metabolism and is evaluating the contribution of non-foliar structures (pods, siliques, silicle) to seed yield and plant productivity. In addition, the lab has collaborated with the Benning lab resulting in a 2023 publication on chloroplast lipids, has a longstanding project that involves the Cousins lab related to C4 metabolism, and works with the Zhang lab on algal projects.

Christoph Benning, MI-ABR described a new retrograde signaling pathway starting with the accumulation of phosphatidic acid in the chloroplast intermembrane space and involving the mediator complex in the nucleus. In addition, he described a new reactive oxygen signaling pathway involving the turnover of a trans fatty acid in the chloroplast mediated by FAD4 and small redox active proteins in Arabidopsis and Chlamydomonas. Collaborators are the Strenkert and Kramer labs at the MSU-DOE Plant Research Laboratory. The work is funded by DOE-BES.  

Nicole Buan NE-AES shared her group’s work to produce the antioxidant and plant growth stimulant, 2-mercaptoethanesulfonate (coenzyme M) in E. coli. In collaboration with Rebecca Roston and Kasia Glowacka, the Nebraska Antioxidant Group has shown CoM application results in increased plant growth by affecting non-photochemical quenching, chloroplast redox balance, hormone signaling, ion homeostasis, and CO₂ fixation although molecular mechanisms are still being defined. The Buan lab designed a synthetic two-gene com^syn operon which converts taurine to CoM via taurine-pyruvate aminotransferase and a proposed ComF CoM synthase enzyme which is hypothesized to catalyze the last step in CoM biosynthesis pathway based on phylogeny, genomics, bioinformatics and protein modeling. A biochemical assay was developed and used to demonstrate time-dependent CoM synthesis in E. coli lysates. Exogenous CoM was shown to be taken up by E. coli and to protect cells from peroxide stress. E. coli com^syn cells were also more resistant to peroxide stress when taurine was supplied. Future work will involve optimizing protein expression and stability and moving the com^syn pathway into plant cells to test the effect of endogenous CoM synthesis on photosynthesis and metabolism.

Asaph Cousins, WA-AES indicated 500 water molecules are lost for every CO2 coming in which results in significant water loss to maintain transpiration stream. Bicarbonate and PEP levels will impact PEPC activity, and bicarbonate is itself affected by carbonic anhydrase. Additionally, how much PEPC or its affinity may play a role as indicated in Flaveria. The goal is to look at factors causing low intracellular CO2 that might be causing a loss in C4 photosynthesis. [HCO3] levels are in part controlled by carbonic anhydrase. The Cousins lab is looking for other ways to shift the curve and influence C4 efficiency.

Felix Fritschi, MO-AES. Current efforts focus on water use efficiency (WUE) at the whole-plant level and relationships with leaf- and canopy level gas exchange and plant hydraulic architecture. Genome wide association analysis in soybean identified novel genetic markers for WUE and biparental mapping revealed several QTL for gas-exchange traits. Additionally, the lab is exploring how breeding for higher yield over the course of nearly 100 years may have inadvertently changed physiological traits in soybean. This effort revealed that public soybean breeders improved soybean WUE, a change that was associated with lower canopy temperatures during reproductive growth as well as with changes in top-soil root architecture and stem xylem vessel characteristics. Ongoing work is examining the contributions of these and other characteristics to the observed changes in WUE.

Katarzyna Glowacka, NE-AES, obtained evidence for a dark accumulation of the zeaxanthin in high chilling tolerance C4 grasses of Miscanthus. Chilling-induced zeaxanthin accumulation in the dark enhanced rate of NPQ induction by 66% in the following morning. The possible mechanisms uncovered here for the unique regulation of NPQ include post-translational regulation of violaxanthin de-epoxidase (VDE), VDE cofactor accessibility, and absence of transcriptional upregulation of zeaxanthin conversion back to violaxanthin. Engineering dark accumulation of zeaxanthin will help improve crop chilling tolerance and promote sustainable production by allowing early spring planting to maximize the use of early-season soil moisture.

Jeff Harper (NV-ARS) obtained evidence for a connection between cellular energy homeostasis and basal concentrations of cytosolic Ca²⁺ ([Ca²⁺]_cyt). When roots are exposed to an external energy source (e.g., sucrose), basal [Ca²⁺]_cyt is reduced. Conversely, when roots are exposed to metabolic inhibitors, basal [Ca²⁺]_cyt increases. This supports a “rules-of-life” model in which changes in basal [Ca²⁺]_cyt either reflect or promote the reprogramming of eukaryotic cells as they adapt to situations that increase or decrease cellular energy.

Jiaxu Li, MS-AES found that overexpression of an abscisic acid-activated protein kinase-like kinas gene can improve soybean photosynthesis and water use efficiency under water limiting conditions. The Li lab isolated dehydration-stimulated peptides from the leaves of rice plants subjected to water deficit. This dehydration-stimulated peptide was also up-regulated by salt stress. This dehydration-stimulated peptide was identified by mass spectrometry-based de novo sequencing as a rapid alkalinization factor. The Li Lab will apply functional analysis of the dehydration-responsive peptide to elucidate its roles in drought adaptation in plants.

Tasios Melis, CA-AES. The work aims to convert fast-growth unicellular cyanobacteria into cell factories for the renewable and carbon-negative generation of high-value bioactive compounds and biopharmaceutical proteins. Cyanobacteria are prokaryotic photosynthetic microorganisms that can generate, in addition to biomass, useful chemicals and proteins / enzymes, essentially from sunlight, carbon dioxide and water. Selected aspects of cyanobacterial production (isoprenoids and high-value proteins / enzymes), and scale-up methods suitable for product generation and downstream processing were addressed in this period. The work focused on the promise of specialty chemicals and proteins production, with isoprenoid products and biopharma proteins as study cases, and the challenges encountered in the expression of recombinant proteins / enzymes, which underline the essence of synthetic biology with these photosynthetic microorganisms. Progress and the current state of the art in these targeted topics were reported. 

Rebecca Roston, NE-AES reported on two projects, the first investigating mechanisms by which lipids are transported to the photosynthetic membrane. Screening through candidates homologous to lipid transport proteins at other membrane contact sites, candidates were identified that disrupt the thylakoid organization in young plastids. Using a membrane fractionation-based approach, candidates that are homologous to mitochondrial cristae organizing proteins were identified. Efforts to measure the effect of these proteins on lipid transport are underway. The second project is a collaborative one with Nicole Buan (NE-AES) and Katarzyna Glowacka (NE-AES), which investigated the mechanisms through which photosynthesis was affected by antioxidant application. Antioxidants increase growth in multiple species and enhance Fv/Fm, while they have different effects on non-photochemical quenching. Two antioxidants were further investigated, Coenzyme M, a small archaeal antioxidant, and ascorbic acid. Results suggest that Coenzyme M directly inhibits violaxanthin de-epoxidase, an enzyme contributing to non-photochemical quenching, while ascorbic acid activates the same enzyme. If we better understood these divergent effects on photosynthesis, we would be better able to use the antioxidants agriculturally. Coenzyme M in particular shows promise as it is small, easily mass-produced, and has different effects than some other antioxidants.

Berkley Walker MI-ABR reported on efforts to determine how plants have acclimated and adapted to photorespiratory flux under elevated temperatures with a goal to develop improvement strategies. This work has demonstrated that the initial reactions of photorespiration appear to uniquely have increased activities in a species (Rhyza stricta) adapted to elevated temperatures, but these activities do not acclimate in Betula papyrifera grown under different photorespiratory pressures. This work has also helped develop strategies for increasing the thermotolerance of photorespiratory enzymes by “learning’ from thermotolerant versions of the enzymes.

Xin Wang FL-AES discussed recent findings in the group that the respiratory Entner-Doudoroff (ED) pathway is incomplete in S. elongatus and likely absent in most cyanobacteria. This was verified by the enzyme kinetics experiments that there are no active enzymes to catalyze 6PG dehydration step in the ED pathway. Instead, KDPG aldolase, another key enzyme in the ED pathway, has an alternative oxaloacetate decarboxylation activity. This alternative enzyme activity is crucial for fine-tuning ATP/NADPH production and consumption under light, ensuring robust growth in cyanobacteria.

Ru Zhang reported the investigation of a putative heat tolerance gene, HTG1, using the unicellular green Alga Chlamydomonas reinhardtii. The HTG1 is highly conserved in green lineage but its function is unknown. HTG1 was identified in the genome-wide pooled screens of Chlamydomonas under high temperatures. The data showed that HTG1 may be localized in the ER under the control condition but migrate to vacuole for degradation under stressful conditions. HTG1 may be also involved in the resume of cell cycle after the heat treatment. The Zhang lab continues to work on understanding the regulation of photosynthesis under abiotic stresses in both green algae and land plants and identify potential targets to improve stress tolerance (especially heat tolerance) in photosynthetic organisms. The Zhang lab has collaborated with Dr. Xin Wang on PSI supercomplexes that is associated with cyclic electron flow and Dr. Doug Allen on algal stress responses.

Business Meeting 

We discussed the future locations to be led by Asaph Cousins with support from Berkley Walker, middle to late October time frame in the Pacific Northwest, followed by Michigan State the following year, and at the next meeting discuss the renewal writing team and future Administrative Advisor who reviews the reports. One possibility is to ask a “Dean”-level person to handle this role. Probably in the year after next there will need to be an effort towards the renewal. Christoph Benning mentioned the mid-term review needs to be handled in a timely fashion. The mid-term review is due in December and would like to base it on the current report. The current grant ends 9/30/27. At the meeting it was discussed to try to continue to seek out new productive members who could participate in future meetings.

Milestones & Activities: 

Objective 1. Identify Strategies to optimize the assembly and function of the photosynthetic membrane.

• The Benning lab made progress towards an understanding of the biosynthesis and function of a central metabolite in lipid biosynthesis in chloroplasts, phosphatidic acid. The role of two phosphatidic acid phosphatases of the chloroplast envelope membrane in chloroplast lipid import was demonstrated. Their loss leads to a strong reduction in growth of the respective Arabidopsis mutant pointing towards a regulatory or signaling role of phosphatidic acid accumulating in the chloroplast intermembrane space. A mutant suppressor screen in this double mutant has yielded a potential target gene encoding a component of the mediator complex.
• The Benning lab linked the biosynthesis of a specific fatty acid (16:1t) found only in a lipid of the photosynthetic membrane to the redox state of the chloroplast in Arabidopsis and now Chlamydomonas. Here, the FAD4 locus encoding the desaturase required for the biosynthesis of a 16:1t overlaps with the gene encoding LCI2, a protein with similarity to the membrane anchor for thylakoid bound ascorbate peroxidase. The abundance of both proteins is regulated by alternative splicing. This discovery led the Benning lab to define a new hypothesis for lipid-based stress sensing in the photosynthetic membrane that is currently being investigated.
• The Roston lab identified multiple proteins as potential contributors to thylakoid membrane homeostasis. Three are implicated by homology to have roles in lipid transport, one in membrane organization. A chloroplast fractionation experiments isolated membranes of intermediate density between the inner envelope membrane and thylakoid membrane, these have unique protein compositions compared to pure inner envelope and thylakoid membranes and represent candidate residents of membrane contact sites between the two membranes. These results have been of broad interest at presentations this year.
• The Kirchhoff lab finalized a quantitative biology pipeline allowing determination of the concentration of energy converting building blocks (photosystems I and II, cytochrome b6f complex, ATP synthase, light harvesting complexes) in plant thylakoid membranes. This complements the existing analytical tools in the lab for photosystem supercomplex distribution, pigments, lipids, and fatty acids. This method was published in 2023. The new pipeline enables new comparative studies between different environmental conditions and mutants, i.e., it allows examination of the plasticity of the photosynthetic hardware in response to environmental cues. Furthermore, the quantitative toolkit forms the foundation for ongoing modeling efforts with the goal of establishing a complete model of thylakoid membranes with molecular resolution.
• Another project in the Kirchhoff lab examined the attachment of the light harvesting complexes attached to both photosystems. A process called state transition that is based on the reversible redistribution of light harvesting complex II from photosystem II to photosystem I. We could show that state transition leads to a perfect balancing of electron transport between the two photosystems. Mechanistically this is triggered by redistribution of certain pool of hyperphosphorylated light harvesting complexes two from stacked to unstacked thylakoid domains. State transition is an important mechanism for regulating energy conversion in plants that contributes significantly to the fitness in the field.

Objective 2. Identify strategies to modify biochemical and regulatory factors that impact the photosynthetic capture and photorespiratory release of CO2.

• The Sharkey lab has been studying the effects of fluctuating light on the carbon metabolism of photosynthesis. In the current year we followed the amount and degree of label in a range of metabolites (metabolite profiling). We found that within 10 seconds of turning off the light nearly all of the carbon in the Calvin-Benson-Bassham (CBB) cycle piled up in 3-phosphoglyceric acid (PGA). PGA can restart the cycle as soon as light is available for making reducing power. Carbon begins to flow into the tricarboxylic acid cycle withing 10 seconds. Over the next few minutes carbon leaves PGA and pyruvate increases. It is not easy for pyruvate to return to the CBB cycle. One pathway for the CBB cycle to restart is the oxidative pentose phosphate, which forms a glucose 6-phosphate (G6P) shunt bypassing the non-oxidative pentose phosphate reactions of the CBB cycle.
• The G6P shunt theoretically could occur in the stroma or the cytosol. When it occurs in the stroma it makes a futile cycle whose benefits are unclear. If it occurs in the cytosol, it can help stabilize the CBB cycle and is one of very few pathways that provide NADPH to the cytosol. A report that the pathway was occurring in the stroma at both high and low CO2 was investigated. The Sharkey lab, using much more direct methods found that the shunt operates only in the cytosol at below ambient, ambient, and three times ambient CO2.
• Labeling of terpenes (e.g. isoprene) follows the same pattern as labeling of CBB intermediates. Whole leaf pyruvate is much less labeled. This was described as a pyruvate paradox; terpene labeling indicated that the pyruvate used for terpene synthesis was heavily labeled but pyruvate in the leaf was much less labeled. We collaborated with Professor Mike Phillips of the University of Toronto to show that the resolution of this paradox is that rubisco makes pyruvate at a rate of just less than 1% of the rate of carboxylations.
• The Cousins lab has used leaf carbon isotope composition (δ13C) to determine how water use efficiency differs in two C4 species of Setaria species and in Sorghum mapping populations. This research helped to better understand the relationship of leaf carbon isotopes and the influence intrinsic water use efficiency to whole plant water use efficiency.
• The group also used δ¹³C and maize genetic diversity to explore biochemical and post-photosynthetic factors that may influence δ¹³ They found that the observed variation in δ¹³C across diverse maize lines is likely driven by differences in CO2 availability and not photosynthetic or respiratory metabolism.
• Little is known about intraspecific variation mesophyll conductance (g_m), which describes the movement of CO2 from the intercellular air spaces to the site of initial carboxylation in the mesophyll, about in C4 plants. To address these questions, g_m was measured by the Cousins Lab on numerous C4 species in response to CO2, employing three different estimates of g_m.  Our results provide strong support for a CO2 response of g_m in Zea mays and indicate that gm in maize is likely driven by anatomical constraints rather than biochemical limitations.  The CO2 response of gm indicates a potential role for CO2-transporting aquaporins in C4- g_m. These results also suggest that water-use efficiency could be enhanced in C4 species such as maize by targeting g_m. 
• If g_m were to limit C4 photosynthesis, it would likely be at low CO2 concentrations (pCO2); however, data on C4- g_m across pCO2 are scarce. The Cousins lab has described the response of C4- g_m to short-term variation in pCO2, at three temperatures in Setaria viridis, and at 25 ºC in Zea mays.  Additionally, the lab has quantified across pCO2 the potential limitations to photosynthesis imposed by stomata, mesophyll and carbonic anhydrase (CA) and the effect of finite g_m calculations of leakiness.  In both species, g_m increased with decreasing pCO2. At pCO2 below ambient, photosynthetic rate was limited by CO2 availability.  In this case, the limitation imposed by mesophyll was similar or slightly lower than stomata limitation.  At very low pCO2, CA further constrained photosynthesis.  High g_m could increase CO2 assimilation at low pCO2 and improve photosynthetic efficiency under situations when CO2 is limited, such as drought.  Finite g_m increased estimates of leakiness over values derived with g_m infinite in Setaria but not in Zea.
• In the Walker Lab, potential limiting steps to photorespiratory carbon processing under elevated temperatures were identified from species adapted to or acclimated to different photorespiratory pressures. Interestingly, while heat-adapted plants showed increased activities of these enzymes, heat-acclimated leaves did not. This work has not been accepted for publication.
• In the Wang Lab, the Entner-Doudoroff (ED) pathway was found to be incomplete in the cyanobacterium elongatus, with its key enzyme KDPG aldolase catalyzing an alternative oxaloacetate decarboxylase reaction. This alternative enzyme activity was found to fine-tune photosynthesis under light. When biosynthesis is excessive under light, an imbalanced ATP/NADPH consumption by amino acids biosynthesis results in feedback inhibition to photosynthesis, exacerbating the problem of carbon shortage. KDPG aldolase is activated to convert oxaloacetate to pyruvate, temporarily bypassing TCA cycle reactions to halt amino acids biosynthesis and allowing light reactions to restore a balanced ATP/NADPH production.

Objective 3. Identify strategies to manipulate photosynthate partitioning.

• In the Allen (MO-ARS) lab, metabolic flux maps were built and refined for Chlamydomonas growing auto- or mixotrophically to understand carbon partitioning. The studies indicated use of glyoxylate cycle without gluconeogenic provision of carbon to other parts of metabolism and describe the contribution of acetate to cellular building blocks. In addition, the lab analyzed the acyl-acyl carrier proteins in cyanobacteria that had been engineered to produce medium chain fatty acids. The work suggests some unexplained lipid/fatty acid remodeling, which is under further investigation and could be of importance for developing crop resilience because membrane lipid remodeling is important to maintaining membrane fluidity in different temperatures. Further, the lab is in the process of crossing soybean lines that have been altered through changes in activity of steps in central carbon metabolism and transgenic tobacco that were engineered to make more lipid in the leaf were studied for responsiveness to heat stress resulting in a submitted manuscript. The work is under revision. Finally, the lab developed a method for acyl-CoA measurement to investigate fatty acid oxidation in oilseed tissues.
• Synechocystis sp. PCC 6803 (Synechocystis) is a unicellular photosynthetic microorganism that is used as a model for photo-biochemical research. It comprises a potential cell factory for the generation of valuable bioactive compounds, therapeutic proteins, and possibly biofuels. A severe limitation in such synthetic biology objectives is the low cell tolerance of heterologous proteins, which are readily degraded by the cellular proteasome. Fusion constructs of recombinant proteins with the light-harvesting CpcA α-subunit or CpcB β-subunit of phycocyanin in Synechocystis have enabled true over-expression of several heterologous isoprenoid biosynthetic pathway enzymes and biopharmaceutical proteins to levels of 10-20% of the total cellular protein. It was shown that cyanobacteria would tolerate heterologous recombinant proteins so long as these are part of a useful to the cell functional complex, which confers advantages in photosynthesis, growth competition, and survival. This period’s work employed the human interferon α-2 protein, as a study case of over-expression and downstream processing. It advanced the state of the art in the fusion constructs for protein overexpression technology by developing the bioresource for target protein separation from the fusion construct and isolation in substantially enriched or pure form. The work brings the cyanobacterial cell factory concept closer to meaningful commercial application for the photosynthetic production of useful recombinant proteins and enzymes.
• The Cushman lab (NV-AES) used the overexpression of a bHLH transcription factor from winegrape to increase the plant biomass and leaf succulence, but increased stomatal conductance, transpiration rate, and instantaneous water-use efficiency, but not reproductive yield or water-deficit, osmotic, or salinity stress tolerance in Nicotiana sylvestris. In contrast, overexpression of this same bHLH transcription factor in soybean resulted in effects similar to those observed in Arabidopsis including increases in leaf thickness and reductions in stomatal density, stomatal conductance, and the rate of leaf water loss during water-deficit stress. Although we have not yet observed significant increases in leaf tissue succulence or leaf thickness, these results show promise for improving water-deficit stress tolerance at the whole plant level.
• The Cushman lab (NV-AES) continued its crassulacean acid metabolism (CAM) engineering work in Arabidopsis. Plants expressing the carboxylation module showed significant increases in rosette diameter and leaf area, dawn/dusk titratable acidity, and malate content. Similarly, plants expressing a core CAM module of 7 genes also showed significant increases in rosette diameter and leaf area, dawn/dusk titratable acidity, and malate content. These results show promise for improving biomass production and water-deficit stress tolerance at the whole plant level.
• The Okita lab had previously demonstrated that while removal of the unique Phosphorylase (Pho1) 80 residue peptide (L80), not present in the mammalian and yeast enzymes, had no effect on the catalytic properties of Pho1, expression of this variant enzyme (Pho1ΔL80) in pho1^- rice line resulted in increased seedling growth, flowering time, biomass, seed weight, and seed yields. To see if the suppressor effects on starch synthesis and photosystem I via Pho1’s interaction with PsaC, the terminal redox protein of photosystem I could be biochemically isolated, rice plants expressing a catalytic-minus Pho1ΔL80 were studied.  Catalytic-minus Pho1ΔL80 exhibited faster growth at the seedling stage and distinct photosystem I properties from wildtype in 1-month plants.  These properties are similar to that exhibited by Pho1ΔL80 plants. The effects on PSI in young plants is consistent with the interaction of catalytic-minus Pho1ΔL80 interacting with a Halo-tagged PsaC as viewed by yeast 2-hybrid analysis.  In 2 months, old plants, however, PSI properties were no different from wildtype likely due to the relative instability of the catalytic-minus Pho1ΔL80 in older tissues.   Catalytic-minus Pho1∆L80 lines were shorter than Pho1∆L80 plant height (120~125 cm) at three months old, flowered about ten to seventeen days later than the wild type and morphology of the seeds produced ranged from shrunken to white-core pseudo-normal. 
• The Buan Lab has created a synthetic two-gene operon to produce the antioxidant plant growth stimulant 2-mercaptoethane sulfonate (Coenzyme M) in coli. New plasmids, E. coli strains and enzyme assays were developed, and a hypothetical gene, MA3299 from the methanogenic archaeon Methanosarcina acetivorans, was shown to encode the elusive ComF coenzyme M synthase enzyme. In collaboration with Rebecca Roston and Kasia Glowacka, one review on exogenous application of antioxidants on plants has been published, two additional manuscripts describing this work have been submitted for publication, four additional manuscripts are in preparation, and two patents have been filed.

Objective 4: Develop strategies to overcome limitations to photosynthetic productivity caused by developmental and environmental factors

• The Below lab (IL – AES) assessed how early planting date influences soybean management practices that should be used to obtain maximum yield efficiently. Row spacing interacted with planting date to affect yield. The May 9 planting yielded more in 76 cm row spacing, but for all the other dates, there was numerically greater yields when grown in narrower 51 cm rows. Fertilizer only modestly increased grain yield, and only significantly for the May 9 and numerically for the May 31 planting date. Fertility was not required for the earliest April 23 planting date, even though it resulted in the highest grain yield.
• The Below lab (IL – AES) discovered that, while a higher rate of nitrogen fertilizer increased corn grain yield, there was no yield gain from split-applying N in a dry growing season, and there was no relationship between hybrid root characteristics and their response to the nitrogen fertilizer level.
• The Below lab (IL – AES) characterized the interactions of multiple agronomic management techniques on soil microbiota and yield of long – term continuous maize.
• The Below lab (IL – AES) performed the first year of a comprehensive survey of trifoliate nutrient levels throughout the season, which influences growth and yield of soybean. We are awaiting the chemical analyses results.
• The Below lab (IL – AES) applied either a living microbial blend or a carbon mixture, which decomposed rye reside at different rates by soybean growth stage R3, but by R7, similar decomposition was achieved. Soybean grain yield was not affected by either of the biological treatments when ammonium thiosulfate (ATS) was added, but when ATS was omitted, grain yield increased numerically.
• The Below lab (IL – AES) discovered that supplying an inoculant mix at planting in combination with low rates of fertilizer (45-135 kg nitrogen ha-1) increased maize vegetative growth, nitrogen accumulation, kernel number, and yield (on average 0.11 Mg ha-1 more yield), and was equal to 12-38 kg nitrogen ha-1 of fertilizer.
• The Harper lab (NV-AES) provided evidence that changes in the basal levels of cytosolic Ca²⁺ can activate a programmed cell death pathway in plants. Insights into how basal levels are controlled are expected to guide future efforts to engineer plants to be more productive under temperature-stress conditions.
• The Harper lab (NV-AES) continues to find evidence for differences in how pollen and vegetative cells sense and respond to heat stress. This is significant because it suggests that strategies to improve heat stress tolerance in whole plants might not be successful in the context of plant reproduction (e., we need to find pollen-specific strategies to improve reproductive stress tolerance).
• The Cushman lab (NV-AES) has continued its screening of the USDA-ARS national cactus pear germplasm collection at the National Arid Land Plant Genetic Resources Unit (NALPGRU). Accurate vegetative (and fruit) biomass production data were obtained for 14 different accessions of cactus pear ( ficus-indica and O. cochenillifera). This three-year study in the Central Valley of California resulted in the identification of a hybrid Opuntia spp. accession PARL 845, hybrid No. 46 (O. ficus-indica x O. lindheimerii), which showed the highest annual mean cladode fresh weight (152.8 Mg ha^-1 year^-1) and cladode dry weight (13.3 Mg ha^-1 year^-1) among all accessions tested. This report is significant because it shows that cactus pear displays great potential as a crop with many uses with lower water inputs than conventional crops for arid and semi-arid environments.
• The Cushman lab (NV-AES) examined the effects of different fertilization rates on four accessions of the USDA-ARS national cactus pear germplasm collection at the NALPGRU. Across these four accessions, cladode fresh weight biomass production improved by 16% and 30% by the application of 50 and 150 kg N ha^-1, respectively. Of the four accessions evaluated, PARL 242 ( cochenillifera) and PARL 582 (Opuntia sp.) showed the best performance.
• The Fritschi lab (MO-AES) has generated a soybean Multi-parent Advanced Generation Inter-Cross (MAGIC) population (~500 lines) developed specifically to study the genetics of water-use efficiency (WUE). The MAGIC population was grown in two field environments, samples were collected for WUE assessment and yield was determined.
• The Fritschi lab (MO-AES) identified novel markers for WUE in a soybean diversity panel and is working with breeders to leverage the markers that were identified for the development of elite germplasm.
• The Fritschi lab (MO-AES) assessed xylem characteristics in obsolete and modern soybean cultivars and determined that breeding for greater yield also caused changes in soybean hydraulic architecture.
• The Walker lab characterized the thermotolerance of the final step of photorespiration, glycerate kinase, from diverse species. This characterization was used to engineer an improved version of this enzyme for Arabidopsis thaliana with broader thermal tolerance. This work has been accepted for publication.
• The Li lab (MS-AES) found that overexpression of an abscisic acid-activated protein kinase-like kinas gene (GmAALK1) can improve soybean photosynthesis and water use efficiency under water limiting conditions. The Li lab isolated dehydration-stimulated peptides from the leaves of rice plants subjected to water deficit. This dehydration-stimulated peptide was also up-regulated by salt stress. This dehydration-stimulated peptide was identified by mass spectrometry-based de novo sequencing as a rapid alkalinization factor.
• The Glowacka lab (NE-AES) showed that soybean events carrying a transgenic allele designed to express the photosystem II subunit S under a tight light regulation, as a means to modulate chloroplast-derived signal for stomata opening, had higher water use efficiency under both well-watered and drought mimic conditions. In replicated rainfed field trials, transgenics displayed up to 24% reduction in water loss per CO2 assimilated, which translated to significantly bigger plants and increase in seed production.
• The Giroux lab (MT-AES) has examined leaf starch and plant productivity in a genome wide association study (GWAS) mapping panel for the second field season. A second year of early and grain fill leaf starch has been recorded and awaits analysis. GWAS will be carried out for all yield traits for two growing seasons, as well as for the combined average of both seasons. In the first year of data for this population, early starch is positively correlated with flowering date (p-value ≤ 0.001) while starch at grain fill is negatively correlated to days to anthesis (p-value ≤ 001). It is expected that analysis for this year’s data will follow the same trends.
• The Giroux lab (MT-AES) built heterologous inbreed family populations, selected from the F4 of a Vida/Dagmar cross that segregate for the single base deletion in Vrn-3D (TraesCS7D02G111600) previously identified as the BobWhite_c5979_731 SNP marker. These populations were grown in a randomized complete block design in the summer of 2024 with data collection on standard agronomic metrics. Flag leaves were collected shortly following anthesis for starch analysis. In this population the non-functional allele delayed heading by 1.5 days (p-value ≤ 0.001), increased yield by 7% (p-value = 0253), but did not significantly impact leaf starch (p-value = 0.2161).

Outputs: See attached list of Publications.

Plans for the Coming Year: 

Objective 1: Identify Strategies to optimize the assembly and function of the photosynthetic membrane.

• The Benning lab is in the process of confirming that accumulation of phosphatidic acid in the chloroplast envelope inter membrane space initiates a signal transduction cascade leading to retrograde signaling. They are also in the process of determining the functional interaction of LCI2 and FAD4 in Chlamydomonas and a manuscript describing this interaction is under revision.
• The Roston lab is in the process of testing the impact of our four candidates of membrane contact sites between the chloroplast inner and thylakoid membranes. We will test their ability to transport lipids and their sub-organellar location. Further, we are preparing a manuscript to publish the screens through which we identified these candidates.
• The Kirchhoff lab is in the process of establishing a quantitative computer-based coarse grain model that will be employed for in-depth mechanistic understanding of structure-function relationships in photosynthetic thylakoid membranes. Furthermore, we will finalize a manuscript about ultrastructural thylakoid dynamics triggered by light.

Objective 2. Identify strategies to modify biochemical and regulatory factors that impact the photosynthetic capture and photorespiratory release of CO₂.

• The Sharkey lab will extend research on stabilizing forces in carbon metabolism during large changes in photosynthetic rate using funding from a DOE grant. These studies will include transient expression of the glucose 6-phosphate transporter and CRISPR-generated knockouts of glucose 6-phosphate dehydrogenases. The Sharkey lab will also collaborate with Amanda Koenig of Jianping Hu's lab at MSU to localize the difference G6P dehydrogenases that are responsible for the G6P shunt.
• The Walker lab will be testing the thermotolerance of plants transformed with more thermostable isoforms of thermotolerant photorespiratory genes.
• The Wang lab will work on dissecting the regulatory mechanism of the KDPG aldolase next to understand how NADP+ increases the oxaloacetate decarboxylation activity to fine-tune photosynthesis in cyanobacteria.

Objective 3. Identify strategies to manipulate photosynthate partitioning.

• Current work in the Allen lab (MO-ARS), includes publishing work related to photosynthesis, metabolic flux and partitioning of photosynthetically assimilated carbon and the consequences of abiotic stress (temperature) on metabolic flow which is currently under investigation.
• The Melis Lab will apply this promising phycocyanin fusion constructs technology to further investigate the basic research aspects of recombinant protein stability and over-expression. The objective would be to compare the over-expression pattern of two different proteins with substantially different amino acid composition and folding patterns. One of them, the fibroblast growth factor 2 (FGF-2) comprises a secondary and tertiary structure of α-helices only, the other, the insulin-like growth factor 1 (IGF-1), comprises a secondary and tertiary structure, of strictly β-sheets. The research will investigate the role of protein folding patterns in the post-translational stability and the role of α-helices versus β-sheets in the recombinant protein accumulation in fusion constructs. Successful commercial application of the fusion constructs method would enable the generation of bioactive compounds and agricultural antigens (oral vaccines) that would alleviate the need to apply excessive amounts of antibiotics in the feed of livestock, poultry, and commercial fisheries, which antibiotics, inevitably, find their way in the human food chain. Heterologous enzyme overexpression will find applications in the generation of bioactive compounds, e.g., plant essential oils, thereby enhancing the value of agriculture and its products.
• The Okita lab will attempt to identify the L80 regulatory elements of Pho1 using a deletion approach. Specifically, the following L80 deletions were constructed: L80∆N (∆1-41), L80∆C (∆42-80), [L80∆M (∆21-59), and L80∆HT (∆1-20, ∆61-80).  Transgenic rice lines expressing these Pho1∆L80 variants have been generated and will be studied in the upcoming year.
• In collaboration with a major Biotech Ag company, maize lines expressing variants Pho1∆L80 have been generated. The study of these maize lines will shed light on whether there are differences in Pho1 function in a C4 environment.
• The Roston lab, in collaboration with the Glowacka and Buan labs, is testing antioxidant application and its effect on carbon partitioning of photosynthate. Two manuscripts are being prepared that describe our photosynthetic experiments in Arabidopsis and tobacco, and a third systems-level analysis of changes in response to antioxidants.
• The Buan lab will pursue enzyme engineering strategies to improve ComF protein expression and stability and will produce plasmids to express the comsyn pathway in plant cells. Submitted manuscripts will be revised as needed, and additional manuscripts will be submitted for publication.

Objective 4. Develop strategies to overcome limitations to photosynthetic productivity caused by developmental and environmental factors.

• The Below lab (IL – AES) will further investigate the role of root architecture and nutrients in corn plant growth and yield.
• The Below lab (IL – AES) will perform a survey of trifoliate nutrient levels throughout the season, which influences growth and yield of soybean.
• The Below lab (IL – AES) plans to investigate the use of cover crops and decomposition techniques to enhance nutrient availability for growing continuous maize.
• The Harper lab intends to determine how changes in basal cytosolic Ca²⁺ concentrations change a plant’s response to the environment.
• The Cushman lab (NV-AES) will continue work on phenotyping soybean plants engineered for increased tissue succulence and water-deficit stress tolerance.
• The Cushman lab (NV-AES) will continue work on phenotyping Arabidopsis and soybean plants engineered for various decarboxylation, carboxylation, and core CAM modules for traits associated with improve biomass production and water-deficit stress tolerance
• The Cushman lab (NV-AES) will complete its assessment of the effects of fertilization rates on cactus pear biomass production under field conditions.
• The Fritschi lab (MO-AES) will continue work on genetic mapping of soybean leaf and leaf photosynthesis traits. Data extraction from the past phenotyping campaign will continue and image-based leaf analysis will be conducted to relate gas exchange data to leaf characteristics.
• The Fritschi lab (MO-AES) will explore relationships between water-use efficiency and hydraulic characteristics in soybeans as influenced by water availability. Experiments will be conducted under field and controlled environment conditions.
• The Fritschi lab (MO-AES) will conduct genome wide association analysis of root-shoot partitioning related traits in a soybean diversity panel.
• The Walker lab will continue characterizing photorespiratory genes from species adapted to high and low temperatures.
• The Li Lab (MS-AES) will apply functional analysis of the dehydration-responsive peptide to elucidate its roles in drought adaptation in plants. The Li Lab will generate overexpression lines of the rapid       alkalinization factor gene in rice and test abiotic stress profiles of gene-overexpressing lines. The Li Lab will generate RNA interference (RNAi) lines for the rapid alkalinization factor gene in rice and test abiotic stress profiles of RNAi lines.
• The Glowacka lab (NE-AES) will explore the effect of different promoters to drive a transgenic allele designed to express the photosystem II subunit S, as a means to modulate chloroplast-derived signal for stomata opening, to achieve improvement of water use efficiency, growth and yield in crops.
• The Giroux lab (MT-AES) prepared a manuscript summarizing results in the RIL population, in which Vrn-3D was identified as a possible candidate behind yield, flowering time, and leaf starch differences. This publication was not previously accepted as more information was requested as to the plausibility of Vrn-3D impacting leaf starch. Changes to the manuscript based on continued field study from summer 2024 have been submitted to Plants where we expect it will be published early 2025.
• The Giroux lab (MT-AES) has selected TILLING mutants for two candidate genes (TraesCS7D02G117800 & TraesCS7D02G111600) linked to Vrn-3D that may explain leaf starch differences. Populations will be developed in hexaploid wheat to determine the impact of knockouts to these genes.
• The Giroux lab (MT-AES) will continue to verify Vrn-3D by repeating field experiments on the Vida/Dagmar HIF population at multiple locations in 2025.
• The Giroux lab (MT-AES) plans to prepare a manuscript based on the GWAS population data in conjunction with multiple location years of analysis on the Vida/Dagmar HIF population. The focus of this manuscript will be on the relationship between flowering time, leaf starch and yield.

Aurand E, Moon, T.S, Buan, N.R., Solomon, K.V., Köpke, M., and EBRC Technical Roadmapping Working Group. 2024. Addressing Climate Change Through Engineering Biology. npj Climate Action 3,9 https://doi.org/10.1038/s44168-023-00089-8.

Cook R, Froehlich JE, Yang Y, Korkmaz I, Kramer DM, Benning C. 2024. Chloroplast phosphatases LPPγ and LPPε1 facilitate conversion of extraplastidic phospholipids to galactolipids. Plant Physiol. 195:1506-1520 doi: 10.1093/plphys/kiae100

Costa Netto, J.R., H.L.T. Almtarfi, J. Li, D.T. Anderson, and F.B. Fritschi. 2024. Soybean water-use efficiency increased over 80 years of breeding. Crop Science. In print.

Davis JA, Poulsen LR, Kjeldgaard B, Moog MW, Brown E, Palmgren M, López-Marqués RL, Harper JF. (2024) Deficiencies in cluster-2 ALA lipid flippases result in salicylic acid-dependent growth reductions. Physiol Plant. 176(2):e14228. doi: 10.1111/ppl.14228.

Evans SE, Xu Y, Bergman ME, Ford SA, Liu Y, Sharkey TD, Phillips MA (2024) Rubisco supplies pyruvate for the 2-C-methyl-D-erythritol-4-phosphate pathway. Nature Plants. doi:10.1038/s41477-024-01791-z 

Fu, X., Walker, B.J. Photorespiratory glycine contributes to photosynthetic induction during low to high light transition. Sci Rep 14, 19365 (2024). https://doi.org/10.1038/s41598-024-70201-3

Gamba D, Lorts CM, Haile A, Sahay S, Lopez L, Xia T, Takou M, Kulesza E, Elango D, Kerby J, Yifru M, Bulafu CE, Wondimu T, Glowacka K, Lasky JR. (2024) The genomics and physiology of abiotic stressors associated with global elevation gradients in Arabidopsis thaliana. New Phytologist https://doi.org/10.1111/nph.20138

Garty Y, Bussi Y, Levin-Zaidman S, Shimoni E, Kirchhoff H, Charuvi D, Nevo R, Reich Z (2024) Thylakoid membrane stacking controls electron transport mode during dark-to-light transition by adjusting the distances between PSI and PSII. Nature Plants 10, 512-524. https://doi.org/10.1038/s41477-024-01628-9

Gregory, L.M., Roze, L.V. & Walker, B.J. (2023) Increased activity of core photorespiratory enzymes and CO₂ transfer conductances are associated with higher and more optimal photosynthetic rates under elevated temperatures in the extremophile Rhazya stricta. Plant, Cell & Environment, 46, 3704–3720. https://doi.org/10.1111/pce.14711

Haupt J, Glowacka K. (2024) Chilling- and dark-regulated photoprotection in an economically important C4 grass. Communications Biology (accepted).

Johnson BS, Allen DK, Bates PD. (2024) “Triacylglycerol stability limits futile cycles and inhibition of carbon capture in oil-accumulating tobacco leaves”. Plant Physiology (accepted).

Kambhampati S, Hubbard AH, Koley S, Gomez JD, Marsolais F, Evans BS, Young JD, Allen DK. (2024) “Stable Isotope Labeled Pathway Elucidation (SIMPEL): using stable isotopes to elucidate dynamics of context specific metabolism”. Communications Biology 7:172-183.

Koley S, Jyoti P, Lingwan M, Allen DK. (2024) “Isotopically Nonstationary Metabolic Flux Analysis (INST-MFA) of Plants: Recent Progress and Future Opportunities”. New Phytologist Tansley Insight 242: 1911–1918.

Li F, Grzybowsky M, Roston RL, Schnable JC. “Nighttime Fluorescence Phenotyping Reduces Environmental Variability for Photosynthetic Traits Enabling the Identification of Candidate Loci in Maize”. Under review by BMC Genomics.

Li, Y., S. Huang, Q. Meng, Z. Li, F.B. Fritschi, and P. Wang. 2024. Pre-silking water deficit induced kernel loss through impaired silk growth and ovary carbohydrate dynamics. Plant-Environment Interactions 5:e10141. DOI: 10.1002/pei3.10141.

Loman MH, Sible CN, Below FE (2024) Soybean planting date affects the relationships between soil test values and grain yield. Soil Science Society of America Journal (in press) https://doi.org/10.1002/saj2.20753

Majhi BK, Melis A (2024) Recombinant protein synthesis and isolation of human interferon alpha-2 in cyanobacteria. Bioresource Technology 400, 130664. https://doi.org/10.1016/j.biortech.2024.130664

Melis A, Hidalgo Martinez D, Betterle N (2023) Perspectives of cyanobacterial cell factories. Photosynth. Res. https://doi.org/10.1007/s11120-023-01056-4

Meng S, Chang SKC, Li J, Puppala N (2024) Identification and protein characterization of peanut lines with relatively lower levels of major allergens. Agricultural Research & Technology: Open Access Journal 28: 556424. https://doi.org/10.19080/ARTOAJ.2024.28.556424

Mengistu M, Cushman JC. (2023) The role of drought-induced proteins regulating drought tolerance in cereals. Tuberosa, R. (ed.). In: Developing drought-resistant cereals, Burleigh Dodds Science Publishing, Cambridge, UK. 4: 117-146. DOI: 10.19103/AS.2022.0109.04

Neupane D, Niechayev A, Petrusa LM, Heinitz C, Cushman JC. (2024) Biomass production potential of 14 accessions of cactus pear (Opuntia spp.) as a food, feed, and biofuel crop for arid lands. Journal of Agronomy and Crop Science. 210: e12704. DOI:10.1111/jac.12705 Cover article.

Oiestad AJ, Blake NK, Tillett BJ, Cook JP, Giroux MJ. (2024) Wheat (Triticum aestivum L.) Leaf Starch During Grain Fill is Linked to Flowering Time and Plant Productivity. Plants, in review.

Renna L, Stefano G, Puggioni MP, Kim SJ, Lavell A, Froehlich JE, Burkart G, Mancuso S, Benning C, Brandizzi F. 2024. ER-associated VAP27-1 and VAP27-3 proteins functionally link the lipid-binding ORP2A at the ER-chloroplast contact sites. Nat Commun. 15:6008. doi: 10.1038/s41467-024-50425-7.

Roston RL, Buan N. Engineering Biology Research Consortium. 2024. Engineering Biology for Space Health. https://roadmap.ebrc.org/engineering-biology-for-space-health/.

Sah SK, Popescu GV, Reddy KR, Klink VP, Li J (2024) The Glycine max abscisic acid-activated protein kinase-like kinase 1 (GmAALK1) modulates drought stress response. Journal of Plant Growth Regulation https://doi.org/10.1007/s00344-024-11287-x

Sahay S, Grzybowski M, Schnable JC, Głowacka K. (2024) Genotype-specific nonphotochemical quenching responses to nitrogen deficit are linked to chlorophyll a to b ratios. Journal of Plant Physiology 297, 154261. https://doi.org/10.1016/j.jplph.2024.154261

Sahay S, Shrestha N, Dias HM, Mural RV, Grzybowski M, Schnable JC, Głowacka K. (2024) Nonphotochemical quenching kinetics GWAS in sorghum identifies genes that may play conserved roles in maize and Arabidopsis thaliana photoprotection. The Plant Journal DOI:10.1111/tpj.16967

Shomo ZD, Li F, Smith CN, Edmonds SR*, Roston RL. 2024. "From Sensing to Acclimation: The Role of Membrane Lipid Remodeling in Plant Responses to Low Temperatures" Plant Physiology, kiae382, https://doi.org/10.1093/plphys/kiae382

Sible CN, Kent AD, Margenot AJ, Below FE (2024) Long-term continuous maize: Impacts on the soil microbiome and implications for residue management. Soil Science Society of America Journal (in press) https://doi.org/10.1002/saj2.20681

Sobańska K, Mokrzycka M, Przewoźnik M, Pniewski T, Budka A, Głowacka K. (2024) Exploring Chilling Stress and Recovery Dynamics in C4 perennial grass of Miscanthus sinensis. PLOS ONE. DOI:10.1371/journal.pone.0308162

Svoboda V, Oung HMO, Koochak H, Yarbrough R, Mckenzie SD, Puthiyaveetil S, Kirchhoff H (2023) Quantification of energy-converting protein complexes in plant thylakoid membranes. Biochim. Biophys. Acta 1864, 148945. doi.org/10.1016/j.bbabio.2022.148945

Sze H, Klodová B, Ward JM, Harper JF, Palanivelu R, Johnson MA, Honys D. (2024) A wave of specific transcript and protein accumulation accompanies pollen dehydration. Plant Physiol. 195(3):1775-1795. doi: 10.1093/plphys/kiae177.

Turc B, Sahay S, Haupt J, Santos TdO, Bai G, Glowacka K. (2024) Up-regulation of non-photochemical quenching improves water use efficiency and reduces whole-plant water consumption under drought in Nicotiana tabacum. Journal of Experimental Botany 75, 3959–3972. https://doi.org/10.1093/jxb/erae113

Woodward LP, Sible CN, Seebauer JR, Below FE (2024) Soil inoculation with nitrogen-fixing bacteria to supplement maize fertilizer need. Agronomy Journal (in press) https://doi.org/10.1002/agj2.21729

Xie N, Sharma C, Rusche K, Wang X. 2024. Phosphoketolase and KDPG aldolase metabolisms modulate photosynthetic carbon yield in cyanobacteria. Plant Cell. (accepted)

Xu C, Shaw T, Choppararu SA, Lu Y, Hudson M, Weekley B, Fisher M, He F, Da Silva Nascimento JR, Wergeles N, Joshi T, Bates P, Koo A, Allen DK, Cahoon E, Thelen J, Xu D. (2024) “FatPlants: A Comprehensive Information System for Lipid-Related Genes and Metabolic Pathways in Plants”. DATABASE baae074:1-10.

Xu Y, Koroma AA, Weise SE, Fu X, Sharkey TD, Shachar-Hill Y (2024) Daylength variation affects growth, photosynthesis, leaf metabolism, partitioning, and metabolic fluxes. Plant Physiol 194:475-490. doi:10.1093/plphys/kiad507

Zhang Y, Kaiser E, Dutta S, Sharkey TD, Marcelis LFM, Li T (2024) Short-term salt stress reduces photosynthetic oscillations under triose phosphate utilization limitation in tomato. Journal of Experimental Botany 75 (10):2994-3008. doi.org/10.1093/jxb/erae089

Zhang NN, Venn B, Bailey C, Xia M, Mattoon EM, Mühlhaus T, Zhang R. Moderate High Temperature is Beneficial or Detrimental Depending on Carbon Availability in the Green Alga Chlamydomonas reinhardtii. Journal of Experimental Botany, 2023. https://pubmed.ncbi.nlm.nih.gov/37877811/

Patents                      NC1200         2024

Hines, C., Roston, R., Erickson, D. and N.R. Buan, 2024. Synthetic operon for the production of 2-mercaptoethane sulfonate (coenzyme M). US patent application 63/659,271.

Rodriguez de Quieroz, A., Brown, J., Vijayan, J., Hines, C. Ramos, N.F., Stone, J.M., Bickford, N. Glowacka, K., N.R. Buan, and R. Roston. 2024. Use of a small, effective antioxidant to increase plant and microbial biomass. US patent application 63/659,175.