Photosynthesis Research

Light quality acclimation is one of the key drivers of cyanobacterial physiology, ecology, and productivity. Although chromatic acclimation type 3 (CA3) is the canonical example of light quality-driven phycobilisome remodeling, full-PAR physiological characterization of CA3 strains has so far been limited. Here, we characterize the spectral acclimation strategies of the cyanobacterium Nostoc sp. CCAP 1453/38 across the full photosynthetically active radiation (PAR) range. Genomic analysis confirmed Nostoc as a CA3 strain capable of dynamically adjusting phycoerythrin (PE) and phycocyanin (PC) content in its phycobilisome (PBS) rods. During cultivation under narrow-band LEDs, PE was upregulated under violet, blue and green light (435-555 nm), optimizing light harvesting primarily in the blue-green part of the PAR spectrum, while PC was upregulated under red light (633-687 nm). Beyond canonical CA3 pigment switching, Nostoc responded to wavelengths poorly absorbed by PBS in two qualitatively different ways. Under growth-constraining blue light (465 nm), the strain upregulated total PBS and photosystem II (PSII) levels and biased phycobilisome coupling toward PSII (an increased PBS-PSII/PBS-PSI ratio). However, this metabolically costly response could not overcome the underlying excitonic imbalance caused by preferential PSI excitation, resulting in a low cell division rate. Under near far-red light (687 nm), PBS-PSII coupling itself was enhanced, yet total PBS content was reduced rather than increased. Specific growth rates remained as high as under red light, suggesting that this PBS-PSII reorganization avoided the metabolic burden of antenna upregulation. These results indicate that the same underlying challenge of PSII under-excitation can trigger qualitatively different acclimation responses, only some of which are energetically affordable. Spectral acclimation thus depends not only on matching pigment composition to incident wavelengths, but also on the metabolic cost of the response. Compared with parallel datasets on CA1 and non-CA strains obtained under identical conditions, our findings extend CA3 characterization beyond the canonical green/red framework, highlight bottlenecks and advantages of light quality acclimation in Nostoc, and provide a physiological basis for optimizing light regimes in controlled cyanobacterial cultivations.

Photosynthetic electron transport is tightly regulated to balance ATP and NADPH production and prevent over-reduction of the photosynthetic machinery. How this balance is maintained under severe ATP limitation, particularly under anoxia, is not fully resolved. Here, we used several deletion mutants of chloroplast ATP synthase subunits from Chlamydomonas reinhardtii to examine how electron flow is reorganized under anoxic conditions through the interplay between linear electron flow, cyclic electron flow, and alternative electron sinks. Using a combination of optical spectroscopy methods and membrane inlet mass spectrometry, we characterized changes in photosynthetic electron transport, proton motive force relaxation and gas exchange. Under oxic conditions, the {Delta}atpH mutant (lacking the c-subunit) showed strong acceptor-side limitation at photosystem I (PSI), whereas atpC1-2 (lacking the {gamma}-subunit) resembled the wild type. Under anoxia, this limitation was partially relieved, coinciding with sustained electron transport and prolonged hydrogen production. In particular, the {Delta}atpH mutant exhibited increased cyclic electron flow under oxic conditions and maintained PSI activity under anoxia despite impaired ATP synthesis. Prolonged hydrogen production was also observed in atpC1-2, which did not share the increased respiration of {Delta}atpH. Hydrogenase activity under anoxia is consistent with a role as an electron sink that may help alleviate PSI acceptor-side limitation and rebalance the redox state of the electron transport chain. These findings show that ATP synthase deficiency reorganizes photosynthetic electron flow and promotes prolonged hydrogen production, and point to hydrogenase as a candidate electron sink under energy-constrained conditions.

Pigment composition and regulation are critical for efficient photosynthesis in the ocean, where light intensity decreases and the spectrum narrows with depth. Fucoxanthin (Fx), the main carotenoid of several microalgal lineages, harvests the blue-green light prevailing in the deep euphotic zone. In pelagophytes, the acylated derivative 19'-butanoyloxyfucoxanthin (19'-BFx) is abundant, yet its function and regulation remain unclear. Here, we investigated how irradiance and spectral quality shape photoacclimation in Pelagomonas calceolata, an abundant and cosmopolitan low-light pelagophyte. Cultures were grown under blue or white light across 2-60 umol photons m^-2 s^-1. We analysed growth, PSII photophysiology, pigment composition, gene expression levels, and the impact of spectral shifts on the 19'-BFx/Fx ratio. Pelagomonas calceolata grew optimally under dim blue light and displayed low non-photochemical quenching (NPQ) under assay conditions. Transcriptomes revealed broad remodelling driven mainly by irradiance and modulated by light colour, including carotenoid-related processes. Dim blue light progressively increased the 19'-BFx/Fx ratio, whereas white light kept it low and reversed the response after a blue-to-white shift. These results identify reversible, spectrum-dependent 19'-BFx accumulation as a key component of photoacclimation in P. calceolata and separate irradiance-driven from spectrum-dependent cellular responses. This plasticity may explain the ecological success of P. calceolata in low-light oceans.

Photosynthetic electron transfer relies on the coordinated function and spatial organization of large protein complexes within the thylakoid membrane. The cytochrome b6f complex (b6f) functionally interconnects photosystem (PS) II and PSI in photosynthetic electron transfer and is equally distributed between appressed and non-appressed thylakoid membranes. Here, we investigate the functional link between the lateral distribution of b6f and efficient photosynthetic electron flow in Chlamydomonas reinhardtii. We engineered strains with stromal fusions between PetA of b6f and two fluorescent proteins (FPs) of different molecular mass: Clover and ATeam. Under oxic conditions, these strains exhibited significantly slower electron transfer rates (ETR), lower PSII quantum yields, and increased donor-side limitation of PSI. State transitions were diminished in the fusion strains, accompanied by a strong impairment of STT7-dependent function, suggesting that the presence of fused FPs at b6f interfere with STT7 function. Yet, ETR phenotypes were STT7-independent and FP fusion did not impact intrinsic b6f function. In situ cryogenic electron tomography revealed a significant depletion of b6f from appressed thylakoid membranes in the ATeam strains, while the overall membrane protein concentration remained unchanged. Overall, our data indicate that a balanced distribution of b6f between appressed and non-appressed thylakoid membranes is essential for regulating photosynthetic electron transfer, highlighting the functional importance of thylakoid molecular architecture in vivo.

Sucrose metabolism is an important feature of the physiology of the green lineage of photosynthetic organisms and has therefore been the subject of considerable research on plants, algae, and cyanobacteria. Canonical sucrose biosynthesis pathways often involve the condensation of NDP-glucose and fructose-6-phosphate through the action of sucrose-phosphate synthase, then the dephosphorylation of sucrose 6-phosphate into sucrose via sucrose-phosphate phosphatase (SPP). However, many cyanobacterial genomes encode multiple homologs of SPP proteins (SPP-like), including variants that appear to lack key residues reported to be important for sucrose 6-phosphate binding. Herein, we examine these SPP-like proteins, focusing on the biochemical and physiological characterization of the SPP-like protein encoded by the cyanobacterial model, Synechococcus elongatus PCC 7942. Bioinformatic analysis suggests that the SPP-like family of proteins is highly conserved across cyanobacterial species and forms distinct phylogenetic clades that are more widely distributed than the canonical SPP proteins themselves. Biochemical analysis of the purified S. elongatus PCC 7942 SPP-like protein reveals that it not only retains the capacity to dephosphorylate sucrose 6-phosphate, but it also may have physiologically relevant phosphatase activity on 3-phosphoglycerate (3-PGA). We provide evidence that the SPP-like family of proteins represents a well-conserved group of phosphatases across cyanobacteria and suggest some enzymes in this family may have evolved a broader substrate specificity relative to the well-characterized SPP family. Our results have broader implications for cyanobacterial regulation of sucrose biosynthesis and other key steps of central carbon metabolism.

Mechanistic interpretation of photosynthetic energy-transfer models requires more than reproduction of experimental observables: a model intended to support mechanistic claims should also respond consistently when its proposed functional organizations are removed. Here, we evaluate whether a calibrated PSI-LHCI transport surrogate encodes physically meaningful principles by applying a 2x2 factorial knockout framework that independently removes site-energy heterogeneity and coupling-strength heterogeneity in a 155-pigment network. All perturbations were evaluated using the same Full-model calibration without parameter refitting. Although the calibrated model reproduced a high excitation-trapping yield, eliminating either energetic or coupling heterogeneity unexpectedly improved its apparent transport performance. A strict zero-coupling control confirmed that coupling itself remained necessary for network-mediated reaction-center access, whereas the supplied organization of coupling strengths was not supported by the surrogate. An audit of the model inputs further identified peripheral localization of all lowest-energy states and effective coupling scales far above those used in structure-based chlorophyll Hamiltonians. These findings do not imply that native PSI favors flat energy landscapes or uniform couplings. Instead, they show that endpoint agreement alone does not validate a mechanistic interpretation of a pigment-network model. Factorial knockout analysis provides a falsification-oriented framework for separating physical necessity from proposed organization, diagnosing numerical compensation, and identifying the constraints required for more predictive models of PSI-LHCI energy transfer.

Chlorophyll fluorescence imaging is a powerful tool used to characterize abiotic and biotic stress response of photosynthesis in plants. However, extracting metrics such as Fv/Fm (maximum quantum yield of photosystem II) from chlorophyll fluorescence images remains a substantial bottleneck in large measurement campaigns, often requiring significant manual effort. To resolve this issue, we developed fvfmPy, a user-friendly program which enables rapid analysis of chlorophyll fluorescence images of arrays of leaf discs. fvfmPy uses algorithmic image segmentation to identify regions of interest and extract fluorescence metrics from tens or hundreds of leaf samples per image with little to no manual input. fvfmPy is developed in Python and includes a graphical user interface enabling intuitive parameter adjustment and real-time quality control checking. We additionally developed fvfmR, an R wrapper package for fvfmPy. Fv/Fm values extracted with fvfmPy were indistinguishable from those obtained using manual methods (r2 > 0.99) but required 94% less time to complete (13 s vs. 200 s per 100 measurements). fvfmPy and fvfmR were built to directly integrate with existing fluorescence software packages and can greatly increase throughput of fluorescence measurements to help address key challenges in agriculture and ecology.

Native polyacrylamide gel electrophoresis is the standard first analytical step after detergent solubilization of thylakoid membranes. It separates photosystem supercomplexes by size and shape, but it forces a polydisperse mixture of protein-detergent particles through a gel of limited pore diameter. Interval-zone free-flow electrophoresis (iZE-FFE) performs that first electrophoretic step in free solution, without ampholytes or immobilines, and returns the complexes as a liquid fraction series ordered by net charge. We describe the operational cycle of iZE, show that bromophenol blue reports the charge density of {beta}-dodecylmaltoside micelles, and apply iZE to Arabidopsis thaliana thylakoid extracts from two sequential solubilization campaigns. After a two-step digitonin then digitonin/{beta}-dodecylmaltoside extraction (experiment 6874), SDS-PAGE of the iZE fractions is crowded: high-molecular mass protein-lipid assemblies of photosystem I and photosystem II release the same subunits into many wells. Native-PAGE of the same fractions resolves ATP-synthase from PSI, a free LHCII pool, anodic PSII, and cathodic PSI, including PSI with and without LHCII. A three-step extraction with a single pH working window (experiment 7154) confirms this charge order. Lowering residual detergent in consecutive extracts still produces partly solubilized high-mass assemblies, whereas a final {beta}-dodecylmaltoside step releases the C2S2M2/C2S2M/C2S2 PSII series and a single PSI band. iZE is therefore a charge-first, orthogonal dimension for native thylakoid biochemistry. Native-PAGE, not SDS-PAGE, is the proper second dimension while solubilization remains incomplete.

Photosynthetic organisms protect themselves from excess light through non-photochemical quenching (NPQ), a process that safely dissipates excess light energy as heat. Current understanding of NPQ is largely drawn from a few model systems, leaving it unclear how universal NPQ mechanisms are across the green lineage. We characterize the slow, sustained NPQ of the green alga, Auxenochlorella protothecoides x symbiontica (UTEX 250-A). The Auxenochlorella genome lacks Light-Harvesting Complex Stress Related (LHCSR), one of the two proteins required for the fast energy-dependent quenching (qE) component of NPQ. Mutants lacking the Photosystem II Subunit S (PSBS)-like gene (psbsl1) in Auxenochlorella had no effect on NPQ, indicating this alga lacks canonical qE. We identified two candidate enzymes associated with zeaxanthin-dependent quenching (qZ), which has slower kinetics: a plant-type Violaxanthin De-Epoxidase (VDE) and an algal-type Chlorophycean Violaxanthin De-Epoxidase (CVDE). High light did not trigger the expected conversion of violaxanthin to zeaxanthin, a pigment change that is a hallmark of qZ. Mutants lacking VDE (vde1), CVDE (cvde1), or both enzymes (vde1 cvde1) still displayed slow, reversible NPQ. However, when the Auxenochlorella VDE1 and CVDE1 were expressed in a VDE-deficient mutant of Nicotiana benthamiana, both enzymes restored zeaxanthin production and NPQ, confirming they are catalytically functional de-epoxidases. Together, these results reveal an alga that retains two functional, evolutionarily distinct xanthophyll-cycle enzymes yet does not rely on typical xanthophyll pigment dynamics for photoprotection. This points to a biological function that is decoupled from de-epoxidase activity, highlighting unexplored diversity in green algal photoprotection.

O_LIThe chloroplast CLP chaperone-protease is essential for chloroplast biogenesis. CLP substrate selection is aided by the N-recognin CLPS1 and CLPF adaptors. They interact with each other and the CLPC1 chaperone, but their specific functions are poorly understood. C_LIO_LIWe employed in vivo CLPC1 substrate-trapping in Arabidopsis by expressing a 35S:CLPC1-TRAP-STREPII transgene in wild-type (WT), clpf, clps1, and clpfclps1 to test the consequences of the loss of these adaptors on CLPC1-trapped proteins. Immunoblotting and protein half-life experiments were carried out for identified CLP substrates. C_LIO_LIExpression of the 35S:CLPC1-TRAP-STREPII in clps1cpf was embryo lethal. CLPF was trapped at a reduced level in clps1, supporting CLPS-CLPF interactions. Chloroplast 1O2 sensor EXECUTER1 (EX1) was trapped in WT and clps1 but not significantly in clpf. Steady-state protein accumulation of EX1 and its homolog EX2 increased 30-fold in the CLPC1-TRAP lines and clpr2-1, but not in clpf or clps1. In planta experiments showed that the half-life of EX1 is [~]3-fold longer in clpc1-1 than in WT, but EX1 half-life was unaffected in clpf. C_LIO_LIWe conclude that the CLP system plays a key role in EX1,2 homeostasis by keeping their intra-chloroplast concentrations low through continuous degradation, upstream of their 1O2 signaling function. C_LI

RationaleRecent viral metagenomic studies have identified a plethora of enzyme-encoding genes in Phycodnaviridae viruses that are not strictly required for viral replication. These enzymes hold an unexpected metabolic potential during the infection process with their specific green algae host. As neither their role in the infection process nor the subcellular localization of these proteins has been experimentally characterized, comparative sequences, structural and biochemical in silico analyses can help generate functional and localization hypotheses. MethodsIn a recent viral metagenomic dataset, we identified a collection of viral homologs involved in bilin biosynthesis: heme oxygenase (vHMOX1) and Phycocyanobilin:Ferredoxin oxidoreductase (vPcyA). Viral and algal homologues were compared through sequence analyses and AlphaFold3 structural predictions. Predicted biochemical properties were analyzed for their compatibility with subcellular compartments. Active site architecture and putative substrate binding were compared between viral and algal proteins using AlphaFold3 and experimentally resolved structures. ResultsViral HMOX1 and PcyA sequences are truncated compared to algal homologs, lacking the N-terminal extension associated with chloroplast targeting. However biochemical properties, including isoelectric point and surface charge distribution, are compatible with localization in chloroplast stroma. Structural comparisons reveal modifications in the viral HMOX1 active site, including partial substrate reorientation and substitutions of key residues, consistent with modified heme-binding properties. In contrast, vPcyA models show no significant differences to their algal counterparts. ConclusionsActive site remodeling in vHMOX1 protein models suggests that these viral homologues may have evolved distinct heme-binding properties. Unlike vPcyA, vHMOX1 homologs appear to have diverged more substantially from their algal counterparts, potentially reflecting functional specialization in the viral infection context. One sentence summary of key findingsOur bioinformatic analyses expand the repertoire of auxiliary metabolic genes in Phycodnaviridae by identifying a conserved heme degradation pathway, non-canonical vHMOX1/PcyA targeting and structural rearrangements surrounding the catalytic sites of viral HMOX1.

Non-photochemical quenching (NPQ) dissipates excess absorbed light energy and protects photosynthetic organisms from photodamage, but its role in regulating the balance between growth, stress tolerance and astaxanthin accumulation in Haematococcus lacustris remains unclear. Here, we investigated how enhanced NPQ affects photosynthetic performance, stress-induced differentiation, and productivity in this astaxanthin-producing microalga. We isolated and characterized an NPQ-enhanced mutant line, A116, using cultivation assays under different stress conditions, analysis of photosynthetic parameters, pigment profiling, and whole-genome resequencing. A116 displayed stronger and faster NPQ induction, driven by increased LHCSR accumulation, resulting in decreased photosynthetic electron transport and lower photochemical efficiency under moderate-to-high light. Enhanced NPQ delayed the transition to astaxanthin-rich cysts under high light, allowing greater biomass accumulation under CO2-limiting conditions. However, under high CO2 availability, where carbon fixation relieved excitation pressure supporting efficient photosynthesis, the enhanced NPQ phenotype reduced growth and astaxanthin productivity compared with the wild type. These results show that NPQ modulates a context-dependent trade-off between photoprotection and productivity in Haematococcus lacustris. Increased energy dissipation can improve high-light tolerance under carbon limitation, but becomes detrimental when absorbed light can be efficiently used for carbon assimilation. Thus, optimal algal productivity requires tuning photoprotective capacity to environmental conditions rather than maximizing NPQ.

Biofilms are widely distributed in both natural and engineered systems and play a fundamental role in microbial ecology and biotechnological applications. When grown on agar substrates, biofilms often exhibit macroscopic structural features due to mechanical instabilities driven by matrix production. These wrinkles encode relevant information about biofilm growth and structural development and have even been shown to possess some functional roles. Despite their relevance, currently available approaches for wrinkle detection from images rely on manual annotation, which is time-consuming, not scalable, and strongly dependent on the annotator. In the present study, we developed an end-to-end machine learning pipeline, named AutoWrinkleID, that starts from bright field images of biofilms and performs wrinkle identification, characterisation, and quantification across multiple bacterial species and strains. The name reflects the automated identification and subsequent characterisation and quantification of biofilm wrinkles. The annotations used to generate the masks, namely images containing only the wrinkle structures, were obtained both through manual labelling and using constitutive fluorescence and motility fluorescence. Interestingly, we found that models trained using only manual annotations perform worse than those trained using motility fluorescence markers. A comparison between motility and constitutive fluorescence masks further indicates that motility based annotations consistently outperform constitutive fluorescence across all strains, strongly suggesting that motile phenotypes are tightly associated with wrinkle structures. Additional computational analysis is conducted on the same dataset to assess the robustness and limitations of the results as a function of training dataset. We also observed that Sholl analysis, originally developed for the study of neuronal dendrites and later applied to wrinkle analysis, can effectively characterize wrinkle patterns, although its applicability is strongly limited by wrinkle shape and by the quality of the masks.

The sea anemone Aiptasia is a model system for understanding cnidarian loss of symbiotic algae under heat stress (bleaching). While Aiptasia polyps have been widely used to study this process, accurate symbiosis phenotyping grapples with discordant length scales: fine spatial resolution (~100 um) is needed across a whole organism (~5 mm). To address this, we consider small (~100 um), optically transparent Aiptasia larvae as a bleaching model suitable for whole-organism phenotyping by fluorescence microscopy with larvae classified as symbiotic when algae are localized within gastrodermal cells. To expedite phenotyping, we introduce a machine-learning (ML) image-analysis pipeline (SYMPHONY) designed for single-larva resolution analysis of intact larvae. SYMPHONY efficiently identifies the cellular location of internalized algae (accuracy: 79%, precision: 82%, recall: 79%, F1 score: 79%; training dataset composed of 1611 total objects). Additionally, SYMPHONY reports statistically significant larval bleaching under heat stress and corroborates manual phenotyping results, while significantly reducing operator labor from hours to minutes. The combination of the Aiptasia larvae model and the SYMPHONY pipeline aims to accelerate our understanding of symbiosis breakdown.

Photoendosymbiosis between the ciliate Paramecium tritobursaria and the green alga Chlorella variabilis provides a model for understanding stable photoendosymbiosis. A defining feature of this association is the perialgal vacuole (PV) membrane, a host-derived membrane that encloses each alga and prevents its digestion. However, the timing of PV membrane maturation remains poorly understood because of the lack of molecular markers to distinguish between immature and mature PV membranes. Previous studies have shown that the establishment of symbiosis proceeds through multiple regulated steps following algal uptake; however, the molecular maturation of the PV membrane has not been directly examined. Here, we report a monoclonal antibody that specifically recognizes the PV membrane in symbiotic P. tritobursaria. Time-course immunofluorescence analysis showed that the PV membrane antigen was absent in the early stages after algal uptake, appeared at 48 h, and was detected in all PV membranes by 72 h. The antigen persisted before and after synchronous PV swelling, an experimentally inducible state associated with the loss of normal PV membrane function, but was absent from the membranes surrounding the digested algae. Our findings provide the first molecular evidence that PV membrane maturation is a temporally regulated checkpoint during the establishment of photoendosymbiosis.

Lipid droplets (LDs) play a central role in cellular carbon and energy homeostasis, in particular through the roles played by numerous LD-associated proteins. A long-standing question concerns how these proteins are molecularly organized at the seemingly overcrowded LD surface. Delayed in TAG Hydrolysis 1 (DTH1) has been identified as a key protein for efficient LD degradation in Chlamydomonas reinhardtii, however the underlying molecular mechanism remains unknown. DTH1 contains several structural features including a coiled-coil domain, a phosphatidylethanolamine (PE) specific lipid-binding domain, intrinsically disordered regions, and an alpha-helical C-terminal domain, that make it a compelling candidate for acting as a molecular scaffold in protein recruitment and/or organelle tethering. In this study, we used the Chlamydomonas knock-out mutant dth1 as an entry point to begin dissecting the molecular organization of proteins at the LD surface. Comparative lipidomic and proteomic analyses of isolated LDs from both WT and dth1 mutants revealed several key features associated with LD degradation during nutrient recovery. First, PE emerged as an important lipid species involved in LD degradation. Second, active communication occurs among multiple subcellular organelles particularly between LDs, peroxisomes and mitochondria. Third, DTH1 was found to play a critical role in remodeling both the LD proteome and lipidome. Furthermore, detailed analyses of LD degradation during nitrogen recovery uncovered the formation of micro-LDs, a phenomenon not yet reported in microalgae. Collectively, these results highlight the tight coordination of metabolic activities across subcellular compartments and emphasize the importance of intracellular carbon trafficking during stress recovery.

Photosynthetic carbon assimilation is sensitive to heat stress, with a key component being the thermal sensitivity of Rubisco activase (RCA). One approach to increasing heat tolerance of photosynthesis is to increase the thermotolerance of RCA. Here, we have used a transgenic approach to express RCA{beta} from Agave tequilana (AtRCA) in Setaria viridis. A second line was created where the endogenous SvRCA{beta} was substituted for AtRCA{beta} through complementation of a null mutant, {triangleup}rcaB (AtRCA[bKO]). In vitro assays showed that Agave RCA readily activates Setaria Rubisco, and that its thermostability is higher than that of Setaria RCA. AtRCA[bKO] plants exhibited higher CO2 assimilation at 25{degrees}C compared to WT and AtRCA plants, although Rubisco content and activation did not change. After six hours of heat stress, AtRCA[bKO] plants retained higher CO2 assimilation rates than AtRCA, however, after 24 hours CO2 assimilation declined to similar levels in all lines, suggesting that RCA may not limit carbon assimilation at the conditions tested. Taken together, our results show that substitution of RCA from a CAM plant into a C4 model allows normal growth and supports slightly increased carbon assimilation under control and short-term heat conditions.

Soybean, a widely cultivated leguminous crop valued for its protein, amino acids, and oil, faces the challenge of maintaining protein levels, which have an inverse correlation with yield. Reducing leaf chlorophyll levels could increase seed protein levels without compromising yield; however, this is yet to be tested. Therefore, to understand the impacts of low chlorophyll mutations on soybean yield and seed composition, we screened and compared 25 low chlorophyll soybean mutants to their 11 dark green parents. PI548210 (Lincoln mutant) demonstrates a higher concentration of protein without affecting yield compared to its dark green parent PI548362 (Lincoln), suggesting it as a good candidate for further large-scale field trials. PI547555 (Y11/y11, Clark mutant) demonstrates a lower concentration of oil without impacting yield, alongside lower gross photosynthesis, but with chlorophyll levels in the pod and seed tissues that are comparable to its dark green parent PI548533 (Clark). These findings are consistent with the oil concentration of the soybean being influenced by pod and seed photosynthesis, which is correlated with pod height and row spacing. Chlorophyll levels in the leaf do not necessarily correlate with those in the pod and seed of low chlorophyll mutants, possibly due to substantially lower expression of chlorophyll synthesis genes in the pod and seed. SIGNIFICANCEO_LIPI548210 (Lincoln mutant), one of twenty-five low chlorophyll soybean mutants, demonstrates a higher concentration of soybean protein without affecting yield compared to its dark green parent (Figure 1 and Table 1). C_LIO_LIPI547555 (Y11/y11, Clark mutant), a low chlorophyll soybean mutant, demonstrates a reduced concentration of soybean oil without impacting yield, alongside lower gross photosynthesis in pod and seed tissues compared to its dark green parent (Figures 3 and Table 2). These findings suggest that the oil concentration of the soybean is influenced by pod and seed photosynthesis, which is in turn influenced by pod height and row spacing (Figure 2). C_LIO_LIChlorophyll levels in the leaf do not necessarily correlate with those in the pod and seed of low chlorophyll mutants, possibly due to substantially lower expression of chlorophyll synthesis genes in the pod and seed (Figure 5-6). C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=84 SRC="FIGDIR/small/744892v1_fig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@4282dcorg.highwire.dtl.DTLVardef@9d565forg.highwire.dtl.DTLVardef@1918292org.highwire.dtl.DTLVardef@1359b1_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO Two low chlorophyll mutants are as healthy as their dark green parents. Lincoln and its low chlorophyll mutant, left; Clark and its low chlorophyll mutant, known as Y11/y11, right. It can be seen by eye that the plants have low chlorophyll (light green/yellow leaves) but a similar growth habit to their dark green parents. See Supplemental Figures 1-4 for contrast, where low chlorophyll mutants are stunted in growth compared to their dark green parents. C_FIG O_TBL View this table: org.highwire.dtl.DTLVardef@657ec9org.highwire.dtl.DTLVardef@166e75borg.highwire.dtl.DTLVardef@df23c7org.highwire.dtl.DTLVardef@1a60124org.highwire.dtl.DTLVardef@194ed96_HPS_FORMAT_FIGEXP M_TBL O_FLOATNOTable 1.C_FLOATNO O_TABLECAPTIONComparison of seed yield, weight, seed composition between low chlorophyll mutants and their dark green parents. ANOVA is used with linear mixed model (random effect = block, fixed effect = variety). Least squares mean is used to compare. For yield and seed composition, N=4 blocks. For leaf chlorophyll (SPAD), N=40. Yield is average yield per plant (g). n.s. = not significant. C_TABLECAPTION C_TBL O_FIG O_LINKSMALLFIG WIDTH=179 HEIGHT=200 SRC="FIGDIR/small/744892v1_fig3.gif" ALT="Figure 3"> View larger version (26K): org.highwire.dtl.DTLVardef@7a368aorg.highwire.dtl.DTLVardef@192b8f0org.highwire.dtl.DTLVardef@1abb738org.highwire.dtl.DTLVardef@89e978_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 3.C_FLOATNO Light response curve of low chlorophyll mutant (Y11/y11, PI547555) and its parent (Clark, PI548533). Rates of net and gross photosynthesis of low chlorophyll (white) and dark green parents (black) pods under field conditions. Each dot represents a value (n=4) {+/-}SE. We assumed that the seeds greatly inhibited the transmittance of light through the pod and used photosynthetic photon flux density for a single-side. C_FIG O_TBL View this table: org.highwire.dtl.DTLVardef@3f0528org.highwire.dtl.DTLVardef@16ba712org.highwire.dtl.DTLVardef@a5ab2aorg.highwire.dtl.DTLVardef@889254org.highwire.dtl.DTLVardef@3efa4f_HPS_FORMAT_FIGEXP M_TBL O_FLOATNOTable 2.C_FLOATNO O_TABLECAPTIONPod photosynthetic parameters for low chlorophyll mutant (Y11/y11, PI547555) and its parent (Clark, PI548533). Photosynthesis was measured 1 September through 15 September 2021 at the University of Illinois Energy Farm in Urbana, IL, USA. The statistical analysis was done using ANOVA with linear mixed model (alpha=0.05). N=4 {+/-} SEM for Clark and N=3 {+/-} SEM for Y11. C_TABLECAPTION C_TBL O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=130 SRC="FIGDIR/small/744892v1_fig2.gif" ALT="Figure 2"> View larger version (23K): org.highwire.dtl.DTLVardef@a36c26org.highwire.dtl.DTLVardef@1116c8forg.highwire.dtl.DTLVardef@ee5e61org.highwire.dtl.DTLVardef@1766712_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 2.C_FLOATNO Low chlorophyll mutant (Y11/y11, PI547555) and its parent (Clark, PI548533) differ in concentration of seed oil, which interacts with height of pod and row spacing. The box plots show the median (central line), the lower and upper quartiles (box) and the minimum and maximum values (whiskers). The statistical analysis was done using ANOVA with linear mixed model (n=3 blocks, alpha=0.05). Least squares mean is used to compare. N.s., non- significant in the analysis. A. Concentration of oil in low chlorophyll mutant seeds from the upper canopy decreased by 4% compared to the dark green parent (18.2% vs 19%) while there was no difference between them in the seeds from the lower canopy (20.2% vs 20.6%). B. Schematic layout of 2013 field setting showing two different row spacings. C. Concentration of oil in low chlorophyll mutant decreased by 2% in 38cm spacing (21.4% vs 22%) while there was no difference in 19cm spacing (21.3% vs 21.7%) in 2013 field. C_FIG O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=162 SRC="FIGDIR/small/744892v1_fig5.gif" ALT="Figure 5"> View larger version (22K): org.highwire.dtl.DTLVardef@68e508org.highwire.dtl.DTLVardef@94a6ccorg.highwire.dtl.DTLVardef@152a187org.highwire.dtl.DTLVardef@1eae137_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 5C_FLOATNO (greenhouse). Correlation between the level of leaf chlorophyll (x-axis: SPAD reading) and the level of immature pod or seed chlorophyll (y-axis, mg/g DW). Line represents the linear regression model. R-squared is a coefficient of determination, the percentage of the response variable variation that is explained by the linear model. Pod is labeled by the fresh weight of seeds it contained. A. Level of chlorophyll of 25-100mg pod (n=18). B. Level of chlorophyll of 100-200mg pod (n=17) . C. Level of chlorophyll of 25-100mg seed (n=17). D. Level of chlorophyll of 100-200mg seed (n=20). C_FIG O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=180 SRC="FIGDIR/small/744892v1_fig6.gif" ALT="Figure 6"> View larger version (28K): org.highwire.dtl.DTLVardef@167fd88org.highwire.dtl.DTLVardef@361472org.highwire.dtl.DTLVardef@786325org.highwire.dtl.DTLVardef@1b53855_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 6.C_FLOATNO Levels of gene expression in chlorophyll synthesis pathway. A. CHL common pathway genes; Glutamyl-tRNA reductase (GluTR). Glutamate 1- semialdehyde aminotransferase (GSA-AT). ALA dehydratase (ALAD). Uroporphyrinogen III synthase (UROS). Uroporphyrinogen III decarboxylase (UROD). Protoporphyrinogen IX oxidase (PPO). B. Mg branch; Mg-chelatase (Mgch). Magnesium-protoporphyrin IX monomethyl ester cyclase (MPEC). Protochlorophyllide reductase (POR). 3,8-divinyl protochlorophyllide a 8-vinyl-reductase (4VCR). Heme pathway; Ferrochelatase (FECH). Heme oxygenase (HO). Phytochromobilin synthase (HY). Data come from Severin et al (2010). RPKM, reads per kilobase per million mapped reads. DAF, days after flowering. The source seed is experimental line A81-356022 which was generated by introgressing G. soja (PI468916) into G. max (A81-356022). C_FIG

Carbon allocation governs how plants partition assimilates among leaves, roots, stems, and reproductive organs, directly shaping growth and yield. Most crop models represent this partitioning using fixed or empirically derived coefficients, which limits their ability to predict how allocation responds to genotype, environment, or their interaction. Eco-evolutionary optimality theory offers an alternative where the allocation strategy is allowed to emerge from the marginal costs and benefits of investing carbon in each organ. While this approach has been applied successfully to trees, it has not previously been developed for herbaceous plants. Here, we present DAESIM2-Plant, a mechanistic plant growth model that couples physiological processes including photosynthesis, stomatal conductance, plant hydraulics, and canopy radiative transfer, with an eco-evolutionary optimal allocation scheme for leaves and roots. This is applied to wheat by coupling it to a source-sink grain production module. Using a series of idealized sensitivity experiments, we show that the model reproduces well-documented patterns of plasticity in carbon allocation: diminishing returns on leaf investment as canopy closure and water limitation are approached, a shift in allocation toward roots under drier conditions, and a root:shoot response that depends jointly on soil moisture and the plant's existing root:leaf balance. Simulating a full growing season across a gradient of soil moisture levels reveals a threshold-like response in canopy development, biomass accumulation, and grain yield, with yield constrained by the same assimilate supply that governs vegetative growth during the critical period and grain filling. This provides a promising basis for generalizing model behaviour across a wide range of environmental conditions. Evaluating these results against experimental and field trial data remains an important next step.

Euglena longa is a heterotrophic model euglenid, of significant research interest as a unicellular eukaryote. To provide broad insight into the localisation of its proteome we used subcellular spatial proteomics (SSP), based on a label-free version of LOPIT-DC (Localisation of Organelle Proteins by Isotope Tagging after Differential Centrifugation), in which organelles are separated by differential centrifugation and each protein is assigned to a compartment from its abundance profile across the fractions. We detected 5,909 proteins, using protein abundance profiles across fractions to resolve 16 different subcellular compartments, such as the plastid, pellicle, and peroxisomes, and assigned high-confidence localisations to 2,955 of them. This study serves as a proteomic dataset to resolve protein compartmentalisation for this euglenid, enabling evolutionary, biological and functional insight and analysis. We are drafting manuscripts concerning the metabolic nature of the plastid, the evolutionary origins of the pellicle, and the functions of the peroxisome. Investigators interested in using this data prior to publication are encouraged to contact us to avoid overlap and to facilitate coordinated and collaborative use of this resource.