NC1200: Regulation of Photosynthetic Processes

(Multistate Research Project)

Status: Active

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SAES-422 Reports

Annual/Termination Reports:

[01/18/2023] [05/06/2024] [11/02/2024]

Date of Annual Report: 01/18/2023

Report Information

Annual Meeting Dates: 11/19/2022 - 11/19/2022
Period the Report Covers: 01/01/2022 - 12/31/2022

Participants

Brief Summary of Minutes

Please see attached file below for NC1200's 2022 annual report.

Minutes Attachment:

2022 Report_NC-1200.pdf

Accomplishments

Publications

Impact Statements

Date of Annual Report: 05/06/2024

Report Information

Annual Meeting Dates: 12/02/2023 - 12/02/2023
Period the Report Covers: 01/01/2023 - 12/31/2023

Participants

Presenters:
Rebecca Roston; Doug Allen: Scott McAdam; Ru Zhang; Asaph Cousins; Kasia Glowacka; Christoph Benning; Won Yim; Xin Wang; Rachael Morgan-Kiss.

Other attendees: Jeffrey Harper; Tasios Melis; Morgan Furze.

Brief Summary of Minutes

Brief Summary of Minutes of Annual Meeting

Scientific presentations were given by all presenters, including three prospective additional members to the Multistate Project (Won Yim; Xin Wang and Rachael Morgan-Kiss). The presentations covered topics outlined in detail below in the annual accomplishments section of the report.

Business Meeting Summary

NC1200 administration. At the 2025 meeting we'll need to organize the next grant, due in 2026. We will also need another Academic Advisor in 2025. It can be an agricultural experimental director. Work for renewal to be initiated in 2026.

Membership. Vote to be held for new members.

NC1200 hosting of past and future meetings. Past meetings 2018 Missou, 2019 Washington, 2020 MSU, 2021 Reno, 2022 Nebraska, 2023 Purdue. Future meetings: 2024 Danforth Center hosted by Ru Zhang and Doug Allen; 2025 MSU hosted by Berkeley Walker (potentially, pending a discussion) with a backup group at Washington hosted by Asaph Cousins & Helmut Kirchoff. Discussion was that in the future timing must occur either before/after the DOE meeting.

Accomplishments

Accomplishments Activities in 2023 are summarized under the different objectives. Objective 1. Identify Strategies to optimize the assembly and function of the photosynthetic membrane.   <ul> <li>The Okita lab (WA-AES) conducted further studies to establish a relationship between the plastidic phosphorylase (Pho1) and PsaC (as well as PsaD), the terminal electron acceptor-donor of photosystem I. Past studies have shown that bacterial expressed PsaC and His-tagged PsaC were insoluble after expression in E. coli, which hindered our subsequent study of protein-protein interactions.  We have now demonstrated that PsaC fused to mCherry and MBP exhibited high expression levels and solubility. Additionally, PsaD fused to HaloTag was well-expressed and successfully purified.</li> </ul>   <ul> <li>The Okita lab (WA-AES) conducted further studies on L80, a negative regulatory 80 residue peptide that is absent in the human and yeast phosphorylase. To identify the proximate location of the negative regulatory sequences, transgenic rice lines harboring selective deletions of the L80 peptide sequences were generated and seeds from T1 plants collected.</li> </ul>   <ul> <li>The Okita lab (WA-AES) applied multiplex CRISPR-Cas12a, which generated two transgenic rice plants carrying long deletions in the L80 region. However, sequence analysis revealed that both had 199-nucleotide deletions near the N-terminus of L80, indicating off-frame deletions occurred.</li> </ul>   <ul> <li>The Okita lab (WA-AES) generated homozygous Pho1 catalytic mutant (Pho1cat-) and Pho1 ∆L80 catalytic mutant (Pho1∆L80cat-) rice lines. Preliminary results indicates that the Pho1cat- and Pho1∆L80cat- lines have lower normal seeds yields similar to the Pho1 knockdown BMF136  indicating that a enzymatically active enzyme is essential for normal starch production in developing seeds. Pho1∆L80cat seedlings, however, had faster growth rates as measured at 14 days after germination (DAG) as Pho1∆L80 compared to wildtype and Pho1cat- These preliminary results suggest that the negative regulatory elements impacting growth function is independent of Pho1 catalytic activity.</li> </ul>   <ul> <li>The Okita lab (WA-AES) identify T2 homozygous lines of Pho1 L80 various deletions: L80∆N [∆1-41], L80∆C[∆42-80]; L80∆M [∆21-59]; and L80∆HT [∆ 1-20, ∆ 61-80]. Preliminary analysis shows that L80∆C has longer panicle length and larger seeds than the three other transgenic genotypes.  These preliminary observations suggest that the C terminal end of the of L80 region has a negative regulatory element in modulating starch synthesis and plant growth.</li> </ul>     Objective 2. Identify strategies to modify biochemical and regulatory factors that impact the photosynthetic capture and photorespiratory release of CO2.   <ul> <li>The Furze Lab (IN-ARS) used a comparative framework along with gas exchange measurements and microCT imaging to examine the drivers of photosynthetic capacity between evergreen and deciduous Quercus (oak) species.</li> </ul>   <ul> <li>This year’s work in the Furze Lab (IN-ARS) showed that deciduous species had higher photosynthetic capacity than evergreen. Their higher photosynthetic capacity was also driven by leaf biochemical and anatomical characteristics. For the latter, deciduous leaves had more densely packed mesophyll, a greater portion of palisade than spongy mesophyll, and a larger mesophyll surface area.</li> </ul>     <ul> <li>This year’s work in the Furze Lab (IN-ARS) work suggests that greater investment in leaf structures such as densely-packed palisade mesophyll facilitates higher photosynthetic capacity in deciduous species and helps compensate for their shorter growing season.</li> </ul>   Objective 3. Identify strategies to manipulate photosynthate partitioning.   <ul> <li>The Giroux lab (MT-AES) analyzed spring wheat leaf starch levels from recombinant inbred lines (RILs) varying for leaf starch. Flag leaves were collected at 14 days after flowering (DAF) over two consecutive field seasons.  Quantitative trait loci (QTL) analysis of RILs identified a single marker (BobWhite_c5970_731) associated with 21-34% of variation for days to flowering, seed yield, leaf starch, and plant biomass in this spring wheat population.  In this population leaf starch had a strong negative correlation with flowering date and plant productivity (p-value ≤ 0.001).</li> </ul>   <ul> <li>Efforts to stably over-express recombinant proteins in cyanobacteria and other photosynthetic systems are hindered due to cellular proteasome activity that efficiently degrades foreign proteins. Recent work from the Melis Lab (CA-AES) showed that a variety of exogenous genes from plants, bacteria, and humans can be successfully and stably over-expressed in cyanobacteria, as fusion constructs with the abundant β-subunit of phycocyanin (the cpcB gene product) in quantities up to 10-15% of the total cell protein.</li> </ul>   <ul> <li>The Melis Lab (CA-AES) found CpcB*P fusion proteins P did not simply accumulate in a soluble free-floating form in the cell but, rather, they assembled as functional (α,β*P)3CpcG1 heterohexameric light-harvesting phycocyanin antenna discs, where α is the CpcA α-subunit of phycocyanin, β*P is the CpcB*P fusion protein, the asterisk denoting fusion, and CpcG1 is the 28.9 kDa phycocyanin disc linker polypeptide.</li> </ul>   <ul> <li>This year the Melis Lab (CA-AES) expanded on this project and showed that the CpcA α-subunit of phycocyanin and the CpcG1 28.9 kDa phycocyanin disc linker polypeptide can also successfully serve as leading sequences in functional heterohexameric (α*P,β)3CpcG1 and (α,β)3CpcG1*P fusion constructs that permit stable recombinant protein over-expression and accumulation.</li> </ul>   <ul> <li>The Melis Lab (CA-AES) found these complexes were shown to form a modified functional phycocyanin light-harvesting antenna and to contribute to photosystem-II photochemistry in the cyanobacterial cells.</li> <li>The Melis Lab (CA-AES) showed that cyanobacterial cells need the assembled phycocyanin for light harvesting, photosynthesis, and survival and, therefore, may tolerate the presence of heterologous recombinant proteins, when the latter are in a fusion construct configuration with phycocyanin in a functional but modified phycobilisome, thus allowing their substantial and stable accumulation.</li> </ul>   <ul> <li>This year the Allen lab (MO-ARS) performed studies on metabolism in moss photosynthesis and carbon and nitrogen metabolism to understand the effects of altered CO2 and nitrogen level on moss growth and productivity. The studies indicated changes in moss development as a result of the altered provisions.</li> </ul>   <ul> <li>The Allen lab (MO-ARS) analyzed the acyl-acyl carrier proteins of mutants developed in the Benning lab (MI-AES) to better understand the lipid membrane metabolism of the chloroplast. Mutants in the lab were developed to detail aspects of chloroplast lipid production. The work, in support of the Benning lab (MI-AES), indicated changes in lipid composition and flux that describe the production of lipid molecular species.</li> </ul>   <ul> <li>The Benning lab (MI-AES) examined soybeans with enhanced levels of malic enzyme that result in production of increased lipid levels in developing seeds. Enhanced malic enzyme in the mitochondria resulted in more significant changes to the free amino acid composition; whereas when extra malic enzyme activity was in the plastid resulted in increases in total lipid. Additionally, changes in fatty acid composition reflected the redistribution of reducing equivalents between organelles that impacts polyunsaturation of fatty acids.</li> </ul>   <ul> <li>In the Allen lab (MO-ARS) transgenic tobacco were engineered to make more lipid in the leaf were studied for responsiveness to heat stress. Stomatal opening and closing was impacted by the presence of lipid droplets in guard cells and resulted in most decrease in crop productivity.</li> </ul>   Objective 4. Develop strategies to overcome limitations to photosynthetic productivity caused by developmental and environmental factors.   <ul> <li>The Giroux lab (MT-AES) analyzed candidate genes associated with the BobWhite_c5979_731 SNP marker and determined that it is a perfect marker for the gene This is the D genome homeologue of Vrn-D3, which is an orthologue of FT in Arabidopsis. Direct sequencing of RIL parents for this gene identified a single base pair deletion in the coding region of Vrn-3D in one of the RIL parents.  Further analysis of individual RILs differing for these alleles observed that plants carrying the deletion flowered later, and were higher yielding under low, moderate, and high nitrogen fertilizer regimes, indicating that this candidate gene is likely responsible for observed differences in flowering, leaf starch, and yield in this population.</li> </ul>   <ul> <li>The Giroux lab (MT-AES) has identified multiple QTLs for flowering date and early leaf starch in the GWAS panel for the first field season.</li> </ul>   <ul> <li>In another project, the Giroux lab (MT-AES) is examining leaf starch and plant productivity in a genome wide association study (GWAS) mapping panel. This trial was planted in two consecutive field seasons and leaf starch collected just prior to heading and at 14 DAF.  The first year of starch extractions has been completed and the second is in process.  In this population, early starch is positively correlated with flowering date yet  (p-value ≤ 0.001) while starch at grain fill is negatively correlated to days to antheis (p-value ≤ 0.001).  Starch at both developmental stages were not significantly correlated to yield. </li> </ul>   <ul> <li>The Glowacka Lab (NE-AES) characterized physiology and growth of the three selected transgenic soybean lines with unregulated NPQ in the growth-chamber conditions. Both under control (field water capacity (FWC) of 90%) and drought (60% FWC) transgenics consumed ~15% less water than wild type. Furthermore, in the desiccation test transgenics’ leaves lose ~20% less water than corresponding wildtype. In control conditions, transgenics had significantly bigger total leaf area what led also to significantly higher fresh leaf area. A much stronger advantage of transgenic modification was seen under drought conditions where not only leaf area and fresh leaf biomass but also the total above ground dry biomass was significantly bigger.</li> </ul>   <ul> <li>The Glowacka Lab (NE-AE) characterized photosynthetic performance of the three selected transgenic soybean lines with unregulated NPQ in the field. In two field trials performed under rain-fed conditions, transgenics had significantly lower stomatal conductance, and significantly higher intrinsic water use efficiency with limited effect on leaf carbon assimilation.</li> </ul>   <ul> <li>The Glowacka Lab (NE-AE) measured growth and yield of the three selected transgenic soybean lines with unregulated NPQ in the field. In 2022 field trail, transgenics had significantly higher dry leaf biomass and significantly higher total weight of seeds and total number of pods.</li> </ul>   <ul> <li>The Below lab (IL – AES) obtained accurate data of soil composition, plant growth, yield, and components on the second year of the multi-year fertilizer trial. The third-year trial using maize (Zea mays ) on the static plots were established, grown, and resulting grain production data was obtained.</li> </ul>   <ul> <li>The Below lab (IL – AES) obtained initial grain yield data in soybean [Glycine max (L.) Merr.] in response to combining planting date and additional agronomic management.</li> </ul>   <ul> <li>The Below lab (IL – AES) characterized the interactions of multiple agronomic management techniques on soil microbiota and yield of long – term continuous maize.</li> </ul>   <ul> <li>The Li lab (MS-AES) has been working on abiotic stresses especially droughtand salinity on photosynthetic productivity of crop plants. We found that silicon application can improve soybean photosynthesis and water use efficiency under water limiting conditions.</li> </ul>   <ul> <li>The Li lab (MS-AES) recently isolated a 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 cleaved carboxyl-terminal peptide of histone H2B. The work showed that histone tail cleavage can be an important molecular response to abiotic stress.</li> </ul>   <ul> <li>The McAdam lab (IN-AES) investigated the role of evaporative demand on the speed of stomatal opening in the light across land plant species, discovering that stomatal opening speed is a function of evaporative demand only in species which have stomata that have mechanical interactions with the epidermis.</li> </ul>   <ul> <li>The McAdam lab (IN-AES) used continuous monitoring of leaf water status and transpiration we have found that an increase in the levels of abscisic acid in the leaf occur at the onset of stomatal closure during drought, which leads to a reduction in photosynthetic rate.</li> </ul>   <ul> <li>The McAdam lab (IN-AES) have found that during a drought, an increase in the levels of ABA in the leaf triggers stomatal closure in Fagus sylvatica - this result resolves a long-standing debate about the mechanism of stomatal regulation in anisohydric species.</li> </ul>   <ul> <li>The McAdam lab (IN-AES) have also found that an increase in ABA levels in the leaf can trigger leaf senescence at the end of the growing season, but that this process is not dependent on ABA.</li> </ul>   <ul> <li>The Harper lab (NV-ARS) collaborated with Jian Hua (Cornell) to provide evidence in Arabidopsis that resting cytosolic Ca2+concentrations are regulated by the combined activities of calmodulin-stimulated Ca2+-pumps in the plasma membrane, vacuole, and endoplasmic reticulum.  Knockout (KO) mutants in these Ca2+-pumps result in salicylic acid (SA)-dependent autoimmunity, which can be suppressed by lowering external supplies of calcium.</li> </ul>   <ul> <li>The Harper lab (NV-ARS) found that knockout (KO) mutants corresponding to vacuolar Ca2+-pumps aca4/11 plasma membrane pumps aca8/10 both show severe hypersensitivities to chilling and heat stress environments.</li> </ul>   <ul> <li>The Harper lab (NV-ARS) created an Arabidopsis lipid flippase mutant harboring a quintuple KO (knockout) of ala8/9/10/11/12. This mutant is 2.2-fold smaller and displays salicylic acid (SA)-dependent autoimmunity, which can be suppressed by lowering external supplies of calcium.</li> <li>The Cushman lab (NV-AES) reported on the developmental dynamics of crassulacean acid metabolism (CAM) in the cactus pear ( ficus-indica) to assess the relative contribution of C3 photosynthesis and CAM in this highly productive and water-use efficient plant species. Developing O. ficus-indica primary and daughter cladodes begin as respiring sink tissues that transition directly to performing CAM once net positive CO2 fixation is observed.</li> </ul>   <ul> <li>The Cushman lab (NV-AES) obtained accurate vegetative (and fruit) biomass production data 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.</li> </ul>   <ul> <li>The Cushman lab (NV-AES) assessed the history, evolutionary, phylogenetic, and biogeographic diversity of tissue succulence in plants, the potential role of this important anatomical adaptive trait to improve the climate resiliency of plants, and the current prospects for engineering tissue succulence to improve salinity and drought tolerance in crops.</li> </ul>   <ul> <li>The Cushman lab (NV-AES) contributed to investigations into 1) the impact of starch biosynthesis in the daytime closure of stomata in the obligate CAM species, Kalanchoe fedtschenkoi, 2) the several different genetic mechanisms responsible for the loss of anthocyanins in betalain-pigmented Caryophyllales species, and 3) the role of epidermal bladder cells as effective barriers against arthropod herbivores rather than contributing only to abiotic stress tolerance.</li> </ul>   <ul> <li>The Cushman lab (NV-AES) continued their phenotypic characterization of Teff (Eragrostis tef), a C4 tropical grass and explored the mechanisms of drought tolerance in this and other cereals within the Poaceae.</li> </ul>   Outputs   See Publications, below.   Plans for Coming Year   Objective 2. Identify strategies to modify biochemical and regulatory factors that impact the photosynthetic capture and photorespiratory release of CO2.   <ul> <li>The Okita laboratory (WA-AES) will continue studies to characterize the interaction of Pho1 with PsaC and PsaD. Bacterial strains harboring expression plasmids for these proteins have been constructed and currently evaluated for co-expression and protein assembly. Alternatively, pull-down studies will be conducted where immobilized recombinant proteins will be incubated with rice extracts and the captured proteins identified by immunoblotting.</li> </ul>   <ul> <li>The Okita laboratory (WA-AES) will continue studies on the negative regulatory L80 peptide by evaluating the growth and photosynthetic properties of rice plants harboring various deletions of the L80 sequences. Similar studies will be conducted with the gene-edited maize plants.</li> </ul> Objective 3. Identify strategies to manipulate photosynthate partitioning.   <ul> <li>In the coming year, the Furze Lab (IN-ARS) will pursue new work related to Objective 3. Identify strategies to manipulate photosynthate partitioning. This work will characterize the seasonal dynamics of nonstructural carbohydrate reserves in ‘ōhi‘a trees and will then manipulate photosynthate partitioning to investigate the role of nonstructural carbohydrate storage and defense investment in ‘ōhi‘a tree resistance to a novel destructive fungal pathogen Rapid ʻŌhiʻa Death.</li> </ul>   <ul> <li>Successful completion of this work in the Furze Lab (IN-ARS) will identify how a tree’s carbon reserves buffer against biotic stress and will inform the development of disease mitigation strategies for Rapid ʻŌhiʻa Death and the management and conservation of Hawaiʻi’s forests.</li> </ul>   <ul> <li>The Giroux lab (MT-AES) prepared a manuscript summarizing results in the RIL population, in which Vrn-3D was identified as the likely candidate behind yield, flowering time, and leaf starch differences.</li> </ul>   <ul> <li>The Giroux lab (MT-AES) will continue to verify Vrn-3D by selecting for HIFs varying for the two alleles in a modern spring wheat population.</li> </ul>   <ul> <li>The Giroux lab (MT-AES) will continue work in the GWAS population. A second year of early and grain fill leaf starch will be analyzed.  GWAS will be carried out for all yield traits for two growing seasons, as well as for the combined average of both seasons. </li> </ul>   <ul> <li>Current work in the Allen lab (MO-ARS), includes completing a manuscript on the response to heat stress of tobacco lines that were engineered to produce high levels of lipids in leaves.</li> </ul>   <ul> <li>In the Allen lab (MO-ARS), analysis of fluxes in pennycress photosynthetic pods will be performed using isotopic labeling and metabolic flux analysis.</li> </ul>   <ul> <li>The Melis Lab (CA-AES) will apply this promising phycocyanin fusion constructs technology to overexpress “oral vaccine”-type proteins that can be used in agriculture and aquaculture to immunize livestock, poultry, and fish, e.g., salmon, in commercial fish farming operations.</li> <li>Successful commercial application of this method by the Melis lab (CA-AES) 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.</li> </ul>   Objective 4. Develop strategies to overcome limitations to photosynthetic productivity caused by developmental and environmental factors.   <ul> <li>The Glowacka Lab (NE-AES) will continue to characterize seed yield from the 2023 field-grown transgenic soybean with NPQ modification.</li> </ul>   <ul> <li>The Glowacka Lab (NE-AES) will perform seeds quality analyses of seeds developed under drought conditions in the transgenic soybean with NPQ modification.</li> </ul>   <ul> <li>The Glowacka Lab (NE-AES) will continue to generate homozygous lines for remaining soybean transgenic events with modified NPQ.</li> </ul>   <ul> <li>The Glowacka Lab (NE-AES) will test the effectiveness of varied promoters in the modification of stoma behavior through NPQ in soybean.</li> </ul>   <ul> <li>The Glowacka Lab (NE-AES) will continue to characterize soybean transgenics with modified NPQ for physiological phenotype under control and stress conditions.</li> </ul>   <ul> <li>The Below lab (IL – AES) will establish and conduct the fourth year of the multi – year fertilization study. Soybean will be grown with a portion receiving the every-year fertilization treatment.</li> </ul>   <ul> <li>The Below lab (IL – AES) will continue to investigate the potential of using agronomic management in combination with planting date to increase the photosynthetic output of yields of soybean.</li> </ul>   <ul> <li>The Below lab (IL – AES) will perform a survey of trifoliate nutrient levels throughout the season, which influences growth and yield of soybean. </li> </ul>   <ul> <li>The Below lab (IL – AES)plans to investigate the use of cover crops and decomposition techniques to enhance nutrient availability for growing continuous maize.</li> </ul>   <ul> <li>The Li Lab (MS-AES) will apply functional analysis of the dehydration-responsive peptide to elucidate their roles in drought adaptation in plants.</li> </ul>   <ul> <li>The Li Lab (MS-AES) will generate overexpression lines of the histone H2B gene in rice and test abiotic stress profiles of gene-overexpressing lines.</li> </ul>   <ul> <li>The Li Lab (MS-AES) will generate RNA interference (RNAi) lines for the histone H2B gene in rice and test abiotic stress profiles of RNAi lines.</li> </ul>   <ul> <li>In the coming year, the McAdam lab (IN-ARS) will continue to investigate the control of stomata by plant hydraulics during drought across land plant species.</li> </ul>   <ul> <li>The McAdam lab (IN-ARS) will also investigate the environmental regulation of stomatal speed across grasses.</li> </ul>   <ul> <li>A key objective for the coming year in the McAdam lab (IN-ARS) is to investigate the evolution of ABA biosynthesis and the role of this in determining stomatal control across land plants.</li> </ul>   <ul> <li>Test candidate genes for their ability to improve heat-stress tolerance in pollen.</li> </ul>   <ul> <li>Determine how changes in resting cytosolic Ca2+concentrations change a plants response to the environment.  </li> </ul>   <ul> <li>Investigate the role of lipid flippases in regulating heat-stress tolerance. </li> </ul>   <ul> <li>The Cushman lab (NV-AES) will continue its life cycle assessment (LCA) and life cycle costing (LCC) analyses related to biomass and bioenergy production from cactus pear ( ficus-indica) in arid and semi-arid climates. We will complete our molecular phylogenetic analysis of the genetic diversity of the national Opuntia spp. germplasm collection in collaboration with the National Arid Land Plant Genetic Resources Unit (USDA-ARS). We will also continue to investigate the causative agents of Opuntia stunting disease and the molecular basis of spine and glochid formation in Opuntia.</li> </ul>   <ul> <li>The Cushman and Yim labs (NV-AES) will continue work on transcriptome and genome sequencing of two obligate CAM species: cochenillifera (diploid) and O. ficus-indica (octoploid).</li> </ul>   <ul> <li>The Cushman lab (NV-AES) will continue its work on investigating the beneficial effects of engineering tissue succulence in soybean (Glycine max).</li> </ul>   <ul> <li>The Cushman lab (NV-AES) will continue its work on installing and optimizing synthetic CAM in thaliana and Glycine max to improve biomass productivity, water-use efficience, and drought tolerance.</li> </ul>   <ul> <li>The Cushman lab (NV-AES) will continue its characterization of the phenotypic diversity within the USDA-ARS germplasm collection of Teff (Eragrostis tef) and in collaboration with the Yim lab (NV-AES) continue the genome and transcriptome analysis of drought-tolerant accessions of tef.</li> </ul>

Publications

Oiestad, A.J., N.K. Blake, B.J. Tillett, J.P. Cook, and M.J. Giroux. Wheat (Triticum aestivum L.) Leaf Starch During Grain Fill is Linked to Flowering Time and Plant Productivity. Crop Science, in review. December 2023.   Glowacka K, Kromdijk J, Salesse-Smith CE, Smith C, Driever SM, Long S. (2023). Is chloroplast size optimal for photosynthetic efficiency? New Phytologist 239: 2197-2211.  <a href="http://doi.org/10.1111/nph.19091">http://doi.org/10.1111/nph.19091</a>   Sahay S, Grzybowski M, Schnable JC, Głowacka K.. (2023) Genetic control of photoprotection and photosystem II operating efficiency in plants. New Phytologist 239: 1068-1082. <a href="https://doi.org/10.1111/nph.18980">https://doi.org/10.1111/nph.18980</a>   Rodrigues de Queiroz A, Hines C, Brown J, Sahay S, Vijayan J, Stone JM, Bickford N, Wuellner M, Glowacka K, Buan NR Roston RL. (2023). The Effects of Exogenously Applied Antioxidants on Plant Growth and Resilience. Phytochemistry Reviews 22: 407-447.  https://doi: 10.1007/s11101-023-09862-3 Sahay S, Shrestha N, Moura Dias H, Mural RV, Grzybowski M, Schnable JC, Glowacka K. <a href="https://doi.org/10.1101/2023.08.29.555201">Comparative GWAS identifies a role for Mendel’s green pea gene in the nonphotochemical quenching kinetics of sorghum, maize, and arabidopsis</a>. bioRxiv doi: 10.1101/2023.08.29.555201 <h3>Kosola KR, Eller MS, Dohleman FG, Olmedo-Pico L, Bernhard B, Winans E, Barten TJ, Brzostowski L, Murphy LR, Gu C, Ralston L, Hall M, Gillespie KM, Mack D, Below FE, Vyn TJ (2023) Short-stature and tall maize hybrids have a similar yield response to split-rate vs. pre-plant N applications, but differ in biomass and nitrogen partitioning. Field Crops Research 295:108880. https://doi.org/10.1016/j.fcr.2023.10880.</h3> B Mohanasundaram, S Koley, DK ALLEN, S Pandey: “Interaction between sugar signaling and nitrogen assimilation controls moss growth in elevated CO2” New Phytologist online Nov (2023).   Y Xu, R Cook, S Kambhampati, S Morley, J Froehlich, DK ALLEN, C Benning: “Arabidopsis Acyl Carrier Protein 4 and Rhomboid Like 10 Act Independently in Chloroplast Phosphatidic Acid Assembly”  Plant Physiology 193(4):2661-2676 (2023).     SA Morley, F Ma, M Alazem, C Frankfater, H Yi, T Burch-Smith, TE Clemente, V Veena, H Nguyen, DK ALLEN: “Expression of malic enzyme reveals subcellular carbon partitioning for storage reserve production in soybeans” New Phytologist 239:1834-1851 (2023).   Hidalgo Martinez D, Melis A (2023) Cyanobacterial phycobilisomes as a platform for the stable production of heterologous enzymes and other proteins. Metabolic Engineering 77:174-187 <a href="https://doi.org/10.1016/j.ymben.2023.04.002">https://doi.org/10.1016/j.ymben.2023.04.002</a>   Momayyezi,M, Prats, K, McElrone, A, Furze, M. (In review) Biochemical and anatomical leaf characteristics of oak trees drive differences in photosynthetic capacity between leaf habits. New Phytologist.   Pichaco Garcia J, Manandhar A, McAdam SAM. 2024. Mechanical advantage makes stomatal opening speed a function of evaporative demand.  Plant Physiology 10.1093/plphys/kiae023.   Mercado-Reyes JA, Soares Pereira T, Manandhar A, Rimer I, McAdam SAM. 2024. Extreme drought can deactivate ABA biosynthesis in embolism resistant species. Plant, Cell and Environment 47: 497-510.   Binstock BR, Manandhar A, McAdam SAM. 2024. Characterizing the breakpoint of stomatal response to vapor pressure deficit in an angiosperm. Plant Physiology 194: 732-740.   Kane CN, McAdam SAM. 2023. Abscisic acid driven stomatal closure during drought in anisohydric Fagus sylvatica. Journal of Plant Hydraulics 9: 002.   McAdam SAM, Manandhar A, Kane CN, Mercado-Reyes JA. 2024. Passive stomatal closure under extreme drought in an angiosperm species. Journal of Experimental Botany <a href="https://doi.org/10.1093/jxb/erad510">10.1093/jxb/erad510</a>.   Li Z, Harper JF, Weigand C, Hua J. (2023) Resting cytosol Ca2+ level is maintained by calmodulin regulated Ca2+ pumps and affects environmental responses in Arabidopsis. Plant Physiology 191(4):2534-2550. doi: 10.1093/plphys/kiad047.   Robichaux KJ, Smith DK, Blea MN, Weigand C, Harper JF, Wallace IS. (2023) Disruption of pollen tube homogalacturonan synthesis relieves pollen tube penetration defects in the Arabidopsis O-FUCOSYLTRANSFERASE1 mutant. Plant Reprod. doi: 10.1007/s00497-023-00468-5. PMID: 37222783   Hurtado-Castano N, Atkins E, Barnes J, Boxall SF, Dever LV, Knerova J, Hartwell J, Cushman JC, Borland AM. (2023) The starch-deficient plastidic PHOSPHOGLUCOMUTASE mutant of the constitutive crassulacean acid metabolisms (CAM) species Kalanchoe fedtschenkoi impacts diel regulation and timing of stomatal CO2 responsiveness. Annals of Botany. DOI: 10.1093/aob/mcad017.   Niechayev NA, Meyer JA, Cushman JC. (2023) Developmental dynamics of crassulacean acid metabolism (CAM) in Opuntia ficus-indica. Annals of Botany. DOI: 10.1093/aob/mcad070   Peréz-Lopéz AV, Lim SD, Cushman JC. (2023) Tissue succulence in plants: Carrying water for climate change. Journal of Plant Physiology. 289: 154081. DOI: 10.1016/j.jplph.2023.154081.   Pucker B, Walker-Hale N, Yim WC, Cushman JC, Crum A, Yang Y, Brockington S. (2023) Evolutionary blocks to anthocyanin accumulation and the loss of an anthocyanin carrier protein in betalain-pigmented Caryophylalles. New Phytologist. In press. DOI: 10.1111/nph.19341.   Moog MW, Yang X, Bendtsen AK, Dong L, Crocoll C, Imamura T, Mori M, Cushman JC, Kant M, Palmgren M. (2023). Epidermal bladder cells as an herbivore defense mechanism. Current Biology. 33: 4662-4673. DOI: 10.1016/j.cub.2023.09.063.   Sage RF, Edwards EJ, Heyduk K, Cushman JC. (2023) Crassulacean acid metabolism (CAM) at the crossroads: Special issue in the Annals of Botany to honor 50 years of CAM research by Klaus Winter. Annals of Botany. DOI: 10.1093/aob/mcad160.   Mengistu M, Cushman JC. (2023) The role of drought-induced proteins regulating drought tolerance in cereals. In: Developing drought resistant cereals. Burleigh-Dodds Scientific Publishing. In press. DOI:   Neupane D, Niechayev NA, Petrusa LM, Heinitz C, Cushman JC. (2023) 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. Submitted. DOI:

Impact Statements

• The Giroux lab (MT-AES) has identified a single gene that may be targeted to improve spring wheat yield. The gene Vrn-3D is a transcription factor involved in the vernalization process and also the photoperiod response pathway- which probably helps to explain differences in leaf starch levels (Objective 3). • Interestingly the Giroux lab (MT-AES) determined that higher leaf starch levels were not beneficial in this population, though the delayed flowering date (2 days) provided a major yield boost in this population (Objective 4). The importance of interactions with Vrn-3D is reported in facultative wheat populations grown in Europe and Asia, but has not received much attention in spring wheat populations. This provides an opportunity to select for improved wheat yield in spring wheat populations by selecting for or against Vrn-3D to improve flowering date and yield at a regional level. • Research from the Giroux lab (MT-AES) into a GWAS spring wheat mapping panel has already identified QTL associated with flowering date as well as early starch level. These may prove to be valuable for targeted breeding for improved photosynthate partitioning. • The long-term goal of this research is to identify ways to increase yield by selecting for improved photosynthesis and/or photosynthate use. The aim of this research is to determine to what degree wheat productivity may be impacted by selecting for increased leaf starch. This in turn would increase productivity and economic return for farmers. • The Glowacka Lab (NE-AES) Interviewed with the reporter which resulted in articles in Nebraska Today, “Refining surge protector in crops could boost yields”. June 5, 2023. https://news.unl.edu/newsrooms/today/article/refining-surge-protector-in-crops-could-boost-yields/ • The Glowacka Lab (NE-AES) Interviewed with the reporter which resulted in articles for Soybean Research & Information Network. June 5, 2023 https://soybeanresearchinfo.com/research-highlight/breeding-soybean-plants-that-lock-in-moisture/ • Under objective 4, the Below lab (IL – AES) made significant progress in characterizing the soil microbiota associated with growing maize continuously long – term. This information provides a basis for producers to grow continuous maize more sustainably based on their field soil type. • Under objective 4, the Below lab (IL – AES) preliminarily determined that early planting of soybean is a management practice that can significantly increase grain yield compared to normal or late planting dates. Other than using later maturity group varieties for the region and providing foliar protection, it does not appear to require any additional management to optimize yield. Conversely, late-planted soybean may require additional management, such as fertility or narrow row spacing to achieve optimal yield, even though the final yield is less than the maximum potential. • Under objective 4, the Below lab (IL – AES) using multi – crop fertilization schemes, preliminary soil data results indicate that fall fertilization prior to the maize season can increase soil nutrient availability at soybean preplant, but it is fertilizer-source dependent. • Under objective 4, the Below lab (IL – AES) found that using either dry-drop or y-drop methods of fertilization significantly increased soil P, K, and S availability at the maize VT growth stage at most soil depths compared to traditional preplant broadcast application. • In-season applications increased soil nutrient availabilities that were not reflected in greater yields, suggesting that, in nutrient-limited conditions, in-season application methods could result in substantial yield increases. • Partitioning of carbon involves central metabolism, possibly the most well-documented set of pathways; however central metabolism is flexible and context specific, differing in species, tissues and responding to inputs from environment. Studies on carbon partitioning and flux outlined here and performed in the Allen lab MO-ARS, were supported through USDA-ARS, NSF, USDA-NIFA and current related work is supported by: M Gehan, DK Allen, PD Bates, H Kirchhoff: USDA-NIFA, “Vegetable oil production in leaves of next generation crops within dynamic environments”. 2021-2023: TP Durrett, DK Allen, V Veena: United Soybean Board/Foundation for Food and Agriculture Research, “An Innovative “Push-Pull-Protect” Approach to Improving Protein Quality”. 2022-2023: JJ Thelen, DK Allen, PD Bates, A Koo, D Xu; NSF/USDA-Plant Genome (PGRP): “Discovering new metabolic constraints and regulatory nodes in oilseeds engineered for enhanced fatty acid synthesis and seed oil” 2018-2022 (no cost extension). • A general guiding principle in the field of biology posits that heterologous gene overexpression in photosynthetic systems is satisfied solely upon the selection of a strong promoter under the control of which to express the desired recombinant protein. • In the vast majority of such eukaryotic gene overexpression efforts in the literature, however, the corresponding target protein cannot be detected in Coomassie-stained SDS-PAGE and its presence, in trace amounts, is evidenced with indirect methods only, such as sensitive Western blot analysis, suggesting that eukaryotic gene expression under the control of a strong promoter does not in fact translate into substantial amounts of the target protein in photosynthetic systems. • This barrier to overexpressing eukaryotic proteins heterologously in photosynthetic tissues is evidenced widely in the literature. • The Melis Lab (CA-AES) contributed with the design of oligonucleotide fusion constructs, as functional protein overexpression vectors in photosynthetic cyanobacteria. • The fusion constructs technology was successfully applied in the overexpression of plant terpene synthases, the human interferon, and the bacterial tetanus toxin fragment C in cyanobacteria. • True overexpression of these heterologous plant, human, and bacterial genes to levels up to 10% of the total cellular protein were demonstrated. • The mechanism and underlying cellular tolerance of the over-expressed recombinant proteins were elucidated and discussed in this year’s work. • Abiotic stresses like drought reduce crop productivity and are likely to become severe problems with the predicted global warming. The intended long-term outcomes of our research are to improve photosynthetic productivity of crop plants under abiotic stress conditions. • Histone H2B tail cleavage has been observed in human cells stressed with the heavy metal nickel. The Li lab showed that histone H2B tail cleavage occurred in rice leaf cells in response to dehydration and salt stress. Our finding suggests that histone H2B tail cleavage can be an important molecular response to abiotic stress in plants. • Histone tail cleavage has been proposed as a novel epigenetic regulatory mechanism for gene expression. Histone H2B tail cleavage could affect gene expression important for abiotic stress responses via changing chromatin structure. • In this year’s work, the Furze Lab (IN-ARS) contributed to understanding biochemical and anatomical influences on the photosynthetic capture of CO2 and the drivers of photosynthetic capacity across the genus Quercus were resolved and discussed in this year’s work. • The work in the McAdam lab (IN-ARS) in 2023 provided novel insight into stomatal regulation and control during drought, particularly the role of evaporative demand in determining stomatal response speeds. This has important implications for modelling canopy responses to changes in light intensity as well as phenotyping for lines that have differences in stomatal response speed. • Considerable insight into the role of ABA on leaf life span and survival of leaves during drought was also gained from the studies conducted in the McAdam lab (IN-ARS). • The Harper lab (NV-ARS) provided evidence that changes in the resting levels of cytosolic calcium correlate with dramatic changes in the transcriptome and a plants reponse to heat and chilling strass. Insights into how resting levels are controlled are expected to guide future efforts to engineer plants to be more productive under temperature stress conditions. • The Harper lab (NV-ARS) continues to find evidence that there are significant difference 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 (i.e., we need to find pollen specific strategies to improve reproductive stress tolerance). • Under objectives 4, the Cushman lab (NV-AES) made significant progress towards engineering tissue succulence in soybean (Glycine max) in collaboration with the Wisconsin Crop Improvement Center. • Under objectives 4, the Cushman lab (NV-AES) made significant progress towards engineering synthetic CAM (SynCAM) both model (A. thaliana) and crop (G. max) species in collaboration with the Wisconsin Crop Improvement Center. These results highlighted the beneficial effects of installation of a carboxylation module gene circuit of CAM for improving biomass production and the effects of installation of a decarboxylation module gene circuit of CAM for improving water-use efficiency and drought tolerance.

Date of Annual Report: 11/02/2024

Report Information

Annual Meeting Dates: 10/26/2024 - 10/26/2024
Period the Report Covers: 11/01/2023 - 10/31/2024

Participants

Allen, Doug (doug.allen@ars.usda.gov) – USDA-ARS/Donald Danforth Plant Science Center; Benning, Christoph (benning@msu.edu) - Michigan State University; Cousins, Asaph (acousins@wsu.edu) – Washington State University; Fritschi, Felix (fritschif@missouri.edu) – University of Missouri; Harper, Jeff (jfharper@unr.edu) – University of Nevada Reno; Melis, Anastasios (melis@berkeley.edu) - University of California, Berkeley; Roston, Rebecca (rroston@unl.edu) – University of Nebraska; Sharkey, Tom (tsharkey@msu.edu) – Michigan State University; Walker, Berkley (berkley@msu.edu) - Michigan State University; Zhang, Ru (rzhang@danforthcenter.org) - Donald Danforth Plant Science Center; Wang, Xin (wangx3@ufl.edu) - University of Florida; Jiaxu Li (JL305@bch.msstate.edu) - Mississippi State University; Giroux, Mike (mgiroux@montana.edu) - Montana State University.
Guests: Burch-Smith, Tessa (tburch-smith@danforthcenter.org) - Donald Danforth Plant Science Center

Brief Summary of Minutes

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⁺]_cyteither 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.

Accomplishments

Milestones & Activities:  Objective 1. Identify Strategies to optimize the assembly and function of the photosynthetic membrane. <ul> <li>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.</li> <li>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.</li> <li>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.</li> <li>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.</li> <li>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.</li> </ul> Objective 2. Identify strategies to modify biochemical and regulatory factors that impact the photosynthetic capture and photorespiratory release of CO2. <ul> <li>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.</li> <li>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.</li> <li>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.</li> <li>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.</li> <li>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.</li> <li>Little is known about intraspecific variation mesophyll conductance (gm), 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, gm was measured by the Cousins Lab on numerous C4 species in response to CO2, employing three different estimates of gm.  Our results provide strong support for a CO2 response of gm 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- gm. These results also suggest that water-use efficiency could be enhanced in C4 species such as maize by targeting gm. </li> <li>If gm were to limit C4 photosynthesis, it would likely be at low CO2 concentrations (pCO2); however, data on C4- gm across pCO2 are scarce. The Cousins lab has described the response of C4- gm 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 gm calculations of leakiness.  In both species, gm 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 gm could increase CO2 assimilation at low pCO2 and improve photosynthetic efficiency under situations when CO2 is limited, such as drought.  Finite gm increased estimates of leakiness over values derived with gm infinite in Setaria but not in Zea.</li> <li>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.</li> <li>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.</li> </ul> Objective 3. Identify strategies to manipulate photosynthate partitioning. <ul> <li>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.</li> <li>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.</li> <li>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.</li> <li>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.</li> <li>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. </li> <li>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.</li> </ul> Objective 4: Develop strategies to overcome limitations to photosynthetic productivity caused by developmental and environmental factors <ul> <li>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.</li> <li>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.</li> <li>The Below lab (IL – AES) characterized the interactions of multiple agronomic management techniques on soil microbiota and yield of long – term continuous maize.</li> <li>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.</li> <li>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.</li> <li>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.</li> <li>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.</li> <li>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).</li> <li>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.</li> <li>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.</li> <li>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.</li> <li>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.</li> <li>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.</li> <li>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.</li> <li>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.</li> <li>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.</li> <li>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.</li> <li>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).</li> </ul> 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. <ul> <li>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.</li> <li>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.</li> <li>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.</li> </ul> Objective 2. Identify strategies to modify biochemical and regulatory factors that impact the photosynthetic capture and photorespiratory release of CO2. <ul> <li>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.</li> <li>The Walker lab will be testing the thermotolerance of plants transformed with more thermostable isoforms of thermotolerant photorespiratory genes.</li> <li>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.</li> </ul> Objective 3. Identify strategies to manipulate photosynthate partitioning. <ul> <li>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.</li> <li>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.</li> <li>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.</li> <li>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.</li> <li>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.</li> <li>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.</li> </ul> Objective 4. Develop strategies to overcome limitations to photosynthetic productivity caused by developmental and environmental factors. <ul> <li>The Below lab (IL – AES) will further investigate the role of root architecture and nutrients in corn plant growth and yield.</li> <li>The Below lab (IL – AES) will perform a survey of trifoliate nutrient levels throughout the season, which influences growth and yield of soybean.</li> <li>The Below lab (IL – AES) plans to investigate the use of cover crops and decomposition techniques to enhance nutrient availability for growing continuous maize.</li> <li>The Harper lab intends to determine how changes in basal cytosolic Ca2+ concentrations change a plant’s response to the environment.</li> <li>The Cushman lab (NV-AES) will continue work on phenotyping soybean plants engineered for increased tissue succulence and water-deficit stress tolerance.</li> <li>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</li> <li>The Cushman lab (NV-AES) will complete its assessment of the effects of fertilization rates on cactus pear biomass production under field conditions.</li> <li>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.</li> <li>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.</li> <li>The Fritschi lab (MO-AES) will conduct genome wide association analysis of root-shoot partitioning related traits in a soybean diversity panel.</li> <li>The Walker lab will continue characterizing photorespiratory genes from species adapted to high and low temperatures.</li> <li>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.</li> <li>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.</li> <li>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.</li> <li>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.</li> <li>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.</li> <li>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.</li> </ul>

Publications

<div>Publications            NC1200         2024</div> <div>Arifuzzaman, M., S. Mamidi, A. Sanz-Saez, H. Zakeri, A. Scaboo, and F.B. Fritschi. 2023. Identification of loci associated with water use efficiency and symbiotic nitrogen fixation in soybean. Frontiers in Plant Science 14:1271849. DOI: 10.3389/fpls2023.1271849</div> 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.

Impact Statements

Objective 1: • The Benning lab (MI AgBioResearch) works under the premise that understanding basic photosynthetic processes and the assembly of the photosynthetic membranes is key to improving crop productivity and enhancing climate resilience in crops. Basic insights gained under the current project have provided new hypotheses that can be tested and new research directions, which led to the successful renewal and initiation of new federally funded projects under which participating scientists at all levels of their careers were trained. Furthermore, synergistic efforts between this project and parallel projects conducted by members of the MSU-DOE Plant Research Laboratory led to multiple collaborative publications. Overall, funds provided under the current umbrella project were successfully leveraged towards accomplishing the mission of the MSU-DOE Plant Research Laboratory and the mission of MSU AgBioResearch. • Rebecca Roston (NE AES) published a review that clarifies the sub-cellular compartmentation of plant lipid changes in response to low temperature stress. 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. Also, the progress on targeting lipid transport to the thylakoid membranes resulted in renewal of support from the Department of Energy, BES “Photosynthetic membrane lipid transport through chloroplast membrane contact site homologs”, 2023 – 2026. • The overarching goal of the Kirchhoff lab is developing a mechanistic understanding of structure-function relationships in plant photosynthetic membranes. The lab covers length scale from sub-nanometer to micrometer. Knowledge from this research is a key element for the identification of strategies to improve crop resilience in changing environmental settings. Research projects in the Kirchhoff lab are supported by grants from the Department of Energy (BES), The National Science Foundation (MCB), and USDA-NIFA.
Objective 2: • Xin Wang has ongoing collaboration with Ru Zhang working on a DOE-funded project to dissect the role of PSI supercomplexes under stress in the psychrophilic algae Chlamydomonas prescuii. Xin Wang joined the NC1200 group last year and work in his group on the KDPG aldolase and its regulation on photosynthesis will likely lead to new findings to help create robust photosynthesis in cyanobacteria. The long-term goal is to translate the knowledge found in cyanobacteria into crop plants to increase photosynthesis and crop yield. This work is funded by an NSF-IOS CAREER grant. X Wang “Glycogen metabolism kick-starts photosynthesis in cyanobacteria” 2021-2026. • The Walker lab receives funding for their work from a new NSF grant, Collaborative Research: Metabolic fluxes from the Calvin-Benson cycle through the parallel shikimate and non-shikimate pathways in plants, NSF-MCB Systems and synthetic biology. This complements additional funding from two other NSF grants and support from the Department of Energy.
Objective 3: • Partitioning of carbon involves central metabolism, possibly the most well-documented set of pathways; however central metabolism is flexible and context specific, differing in species, tissues and responding to inputs from environment. Studies on carbon partitioning and flux outlined here and performed in the Allen lab MO-ARS, were supported through USDA-ARS, NSF, USDA-NIFA and Department of Energy: M Gehan, DK Allen, PD Bates, H Kirchhoff: USDA-NIFA, “Vegetable oil production in leaves of next generation crops within dynamic environments”. 2021-2023 (no cost extension), EB Cahoon, DK Allen, PD Bates, TP Durrett, JM Fox, MA Gehan, T Joshi, C Lu, MJ Smanski, JJ Thelen, R Welti, D Xu: Department of Energy, “B5: Bigger Better Brassicaceae Biofuels and Bioproducts”. 2022-2027. • Work by the Melis Lab (CA-AES) has multiple impacts, as itemized below: A general guiding principle in the field of biology posits that heterologous gene overexpression in photosynthetic systems is satisfied solely upon the selection of a strong promoter under the control of which to express the desired recombinant protein. In the vast majority of such eukaryotic gene overexpression efforts in the literature, however, the corresponding target protein cannot be detected in Coomassie-stained SDS-PAGE and its presence, in trace steady-state amounts, is evidenced with indirect methods only, such as sensitive Western blot analysis, suggesting that eukaryotic gene expression under the control of a strong promoter does not in fact translate into substantial amounts of the target protein in photosynthetic systems. This barrier in the overexpression of heterologous eukaryotic proteins in photosynthetic tissues is evidenced widely in the literature. The Melis Lab contributed with the design of oligonucleotide fusion constructs, as functional protein overexpression vectors in photosynthetic cyanobacteria. The fusion constructs technology was successfully applied in the overexpression of plant terpene synthases, the human interferon, and the bacterial tetanus toxin fragment C in cyanobacteria. True overexpression of these plant, human, and bacterial origin genes to levels up to 10% of the total cellular protein were demonstrated. The mechanism and underlying cellular tolerance of the over-expressed recombinant proteins will be further investigated in the coming period. • Rebecca Roston (NE), Nicole Buan (NE) and Kasia Glowacka (NE), and have published two patents describing the application of Coenzyme M to plants, and the production of it in bacteria. 2020-077 Application number, 63/659,175. Use of a small, effective antioxidant to increase plant and microbial biomass. 2024-006 Application number 63/659,271 Synthetic operon for the production of 2-mercaptoethane sulfonate (coenzyme M). • Nicole Buan (NE) is a Council Member of the Engineering Biology Research Consortium and served on the Leadership Team for the Engineering Biology for Space Health research roadmap. The EBRC identifies critical research needs in engineering biology to promote a sustainable bioeconomy and advises the US government on strategic research objectives. Nicole Buan (NE) organized a Synthetic Biology for Sustainability and Resilience research and collaboration workshop to identify research strengths and opportunities for collaboration in the Nebraska jurisdiction. • The Giroux lab (MT-AES) continued characterization of a single gene that can be targeted to improve spring wheat yield. The gene Vrn-3D is a transcription factor involved in bringing together the vernalization process, the photoperiod response pathway, and the circadian oscillation response to initiate flowering. Significant changes to this developmental process help to explain differences in leaf starch levels.
Objective 4: · The Below lab (IL – AES) characterized the soil microbiota associated with growing maize continuously long – term and published the findings. This information provides a basis for producers to grow continuous maize more sustainably based on their field soil type. · The Below lab (IL – AES) concluded that early-planted soybean can grow large enough to take advantage of the long early-summer days and optimize yield with minimal fertilizer input. In contrast, late-planted soybean typically yields less, and may need some fertilizer to achieve the maximum yield. · The Below lab (IL – AES) discovered that commercial corn hybrids exhibited considerable variation in their root architecture, and differences in root characteristics can affect the utilization of nitrogen fertilizer to foster photosynthesis and yield. • The Below lab (IL – AES) found that using ammonium thiosulfate with termination treatments mitigated the soybean yield penalty typically arising from cereal rye cover crops. • The Below lab (IL – AES) concluded that a nitrogen-fixing inoculant mix added to the nitrogen fertilizer, for small, but significant, increases in maize plant growth and yield. • The Harper lab created a genetically encoded ratio-metric ATP sensor by fusing a MaLion-Red ATP sensor to a Neon-Green normalization reference. The ratio-metric feature allows the status of cellular ATP concentrations to be compared between different cell types. This reporter is being used to evaluate the connection between basal levels of calcium and the regulation of energy homeostasis. • Research efforts by the Cushman lab (NV-AES) for tissue succulence engineering represent an innovative strategy for improving biomass and reproductive yields and water-use efficiency in soybean with potential applications to other crops. • The Cushman lab (NV-AES) made significant progress towards engineering synthetic CAM (SynCAM) in the model species (A. thaliana) and crop (G. max) species in collaboration with the Wisconsin Crop Improvement Center. Installation of a carboxylation module and core CAM gene circuits can be used to increase biomass production, whereas the installation of a decarboxylation module gene circuit can be used to improve water-use efficiency and drought tolerance. • The screening and evaluations of cactus pear accessions by the Cushman lab (NV-AES) will have positive impacts on our understanding of expected biomass yields of elite Opuntia accessions under various conditions within the continental USA and will allow more precise estimates of their bioenergy and carbon sequestration potential. • The Fritschi lab (MO-AES) continues to study drought and high temperature effects on plants as they are the most important factors limiting crop yields around the world. In Missouri, drought is the most common reason for crop insurance payments to farmers. Thus, development of more drought and heat tolerant soybean varieties is critical for US and Missouri soybean farmers. We identified genetic markers for carbon isotope discrimination and leaf gas exchange traits. This information is being leveraged in collaboration with public soybean breeders to develop more drought tolerant germplasm. Additionally, we discovered that water use efficiency increased over the course of 80 years of soybean breeding. At the same time, mid-day canopy temperatures during reproductive development decrease with the year of cultivar of release. The examination of past, inadvertent changes associated with breeding for yield provides insights about potential targets for continued germplasm improvement in soybean. Given the impact of drought and heat stress on soybean yields and the climate conditions predicted in the future; this research is of great importance for U.S. soybean farmers. This research also complements ongoing projects funded by the United Soybean Board as well as the Missouri Soybean Merchandising Council. • The Zhang lab (Donald Danforth Plant Science Center) studies how photosynthetic cells respond to high temperatures by using both green algae and land plants as models. Our ultimate goal is to engineer more efficient and robust photosynthesis under high temperatures for improved agricultural production and biomass accumulation. We investigated the dynamics of heat-induced cyclic electron flow (CEF) around photosystem I (PSI) in the model green alga Chlamydomonas reinhardtii under moderate and acute high temperatures. In collaboration with the Morgan-Kiss lab, we also investigated CEF in both psychrophilic and mesophilic Chlamydomonas species. We are currently investigating heat effects on thylakoid structures in Chlamydomonas reinhardtii by using multiscale Cryo-Volume Electron Microscopy and Cellular Cryo-Electron Tomography. Additionally, we generated transgenic mutants with altered photoprotection (non-photochemical quenching, NPQ) in the C4 model plant Setaria viridis and investigated the regulation of NPQ in C4 model plants. These results help us understand the regulation of C4 photosynthesis and provide insights for improving photosynthesis in C4 crops. • The Li Lab (MS-AES) continues to study drought and salinity effects on crop plants. Abiotic stresses like drought reduce crop productivity and are likely to become severe problems with predicted global warming. The intended long-term outcomes of our research are to improve photosynthetic productivity of crop plants under abiotic stress conditions. Rapid alkalinization factor peptides have been implicated in plant responses to biotic as well as abiotic stress. The Li lab showed that a rapid alkalinization factor in rice is upregulated in response to dehydration and salt stress. Our finding suggests that the rice rapid alkalinization factor may serve as an important signaling molecule for abiotic stress responses. • The Glowacka lab (NE-AES) studies resistance of photosynthesis to abiotic stresses. Our ultimate goal is to engineer more efficient and robust crops for food, feed and biomass. Studies on resistance of photosynthesis outlined here and performed in the Glowacka lab NE-ARS, were supported through NSF CAREER grant “CAREER: Understanding non-photochemical quenching under chilling in the warm-season C4 grasses”, Award OIA-2142993, (2022-2027), $1,375,334. The progress which we made on engineering the better water use efficiency allowed us to secure more founding by new project from Nebraska Soybean Board, “More soybean for less water: genetic approach for improving water use efficiency”, (2024-2025), $46,407. • Interestingly the Giroux lab (MT-AES) determined no significant difference in leaf starch for the Dagmar/Vida HIF population, though the delayed flowering date (1.5 days) provided a major yield boost in this population. The importance of interactions with Vrn-3D is reported in facultative wheat populations grown in Europe and Asia but has not received much attention in Montana adapted spring wheat populations. This provides an opportunity to select for improved wheat yield in spring wheat populations by selecting for or against Vrn-3D to improve flowering date and yield at a regional level. • Research from the Giroux lab (MT-AES) into a GWAS spring wheat mapping panel has already identified QTL associated with flowering date as well as early starch level. These QTL are being refined with a second year of location data which will provide valuable information for targeted breeding for improved photosynthate partitioning. The long-term goal of this research is to identify ways to increase yield by selecting for improved photosynthesis and/or photosynthate use. The aim of this research is to determine to what degree wheat productivity may be impacted by selecting for increased leaf starch. This in turn would increase productivity and economic return for farmers.