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We highlight the achievements of four pioneering Black plant scientists to raise awareness of the importance of diversity, equity, and inclusion in science. Their stories come alive at Historically Black Colleges and Universities through exhibits of science and art and classroom activities (https://www.plantcellatlas.org/pca-art-exhibit.html).

To understand and engineer plant metabolism, we need a comprehensive and accurate annotation of all metabolic information across plant species. As a step towards this goal, we generated genome-scale metabolic pathway databases of 126 algal and plant genomes, ranging from model organisms to crops to medicinal plants (https://plantcyc.org). Of these, 104 have not been reported before. We systematically evaluated the quality of the databases, which revealed that our semi-automated validation pipeline dramatically improves the quality. We then compared the metabolic content across the 126 organisms using multiple correspondence analysis and found that Brassicaceae, Poaceae, and Chlorophyta appeared as metabolically distinct groups. To demonstrate the utility of this resource, we used recently published sorghum transcriptomics data to discover previously unreported trends of metabolism underlying drought tolerance. We also used single-cell transcriptomics data from the Arabidopsis root to infer cell-type specific metabolic pathways. This work shows the quality and quantity of our resource and demonstrates its wide-ranging utility in integrating metabolism with other areas of plant biology. This article is protected by copyright. All rights reserved.

Plant metabolism is a pillar of our ecosystem, food security, and economy. To understand and engineer plant metabolism, we first need a comprehensive and accurate annotation of all metabolic information across plant species. As a step towards this goal, we previously created the Plant Metabolic Network (PMN), an online resource of curated and computationally predicted information about the enzymes, compounds, reactions, and pathways that make up plant metabolism. Here we report PMN 15, which contains genome-scale metabolic pathway databases of 126 algal and plant genomes, ranging from model organisms to crops to medicinal plants, and new tools for analyzing and viewing metabolism information across species and integrating omics data in a metabolic context. We systematically evaluated the quality of the databases, which revealed that our semi-automated validation pipeline dramatically improves the quality. We then compared the metabolic content across the 126 organisms using multiple correspondence analysis and found that Brassicaceae, Poaceae, and Chlorophyta appeared as metabolically distinct groups. To demonstrate the utility of this resource, we used recently published sorghum transcriptomics data to discover previously unreported trends of metabolism underlying drought tolerance. We also used single-cell transcriptomics data from the Arabidopsis root to infer cell-type specific metabolic pathways. This work shows the continued growth and refinement of the PMN resource and demonstrates its wide-ranging utility in integrating metabolism with other areas of plant biology.One-sentence SummaryThe Plant Metabolic Network is a collection of databases containing experimentally-supported and predicted information about plant metabolism spanning many species.

With growing populations and pressing environmental problems, future economies will be increasingly plant-based. Now is the time to reimagine plant science as a critical component of fundamental science, agriculture, environmental stewardship, energy, technology and healthcare. This effort requires a conceptual and technological framework to identify and map all cell types, and to comprehensively annotate the localization and organization of molecules at cellular and tissue levels. This framework, called the Plant Cell Atlas (PCA), will be critical for understanding and engineering plant development, physiology and environmental responses. A workshop was convened to discuss the purpose and utility of such an initiative, resulting in a roadmap that acknowledges the current knowledge gaps and technical challenges, and underscores how the PCA initiative can help to overcome them., National Science Foundation 1916797 David W Ehrhardt, Kenneth D Birnbaum, Seung Yon Rhee; National Science Foundation 2052590 Seung Yon Rhee

Summary Adult mesenchymal stem cells, including preadipocytes, possess a cellular sensory organelle called the primary cilium. Ciliated preadipocytes abundantly populate perivascular compartments in fat and are activated by a high-fat diet. Here, we sought to understand whether preadipocytes use their cilia to sense and respond to external cues to remodel white adipose tissue. Abolishing preadipocyte cilia in mice severely impairs white adipose tissue expansion. We discover that TULP3-dependent ciliary localization of the omega-3 fatty acid receptor FFAR4/GPR120 promotes adipogenesis. FFAR4 agonists and ω-3 fatty acids, but not saturated fatty acids, trigger mitosis and adipogenesis by rapidly activating cAMP production inside cilia. Ciliary cAMP activates EPAC signaling, CTCF-dependent chromatin remodeling, and transcriptional activation of PPARγ and CEBPα to initiate adipogenesis. We propose that dietary ω-3 fatty acids selectively drive expansion of adipocyte numbers to produce new fat cells and store saturated fatty acids, enabling homeostasis of healthy fat tissue.

In addition to its well-known roles as an electrophile and general acid, the side chain of histidine often serves as a hydrogen bond (H-bond) acceptor. These H-bonds provide a convenient pH-dependent switch for local structure and functional motifs. In hundreds of instances, a histidine caps the N-terminus of α- and 310-helices by forming a backbone NH···Nδ1 H-bond. To characterize the resilience and dynamics of the histidine cap, we measured the trans H-bond scalar coupling constant, (2h)JNN, in several forms of Group 1 truncated hemoglobins and cytochrome b5. The set of 19 measured (2h)JNN values were between 4.0 and 5.4 Hz, generally smaller than in nucleic acids (~6-10 Hz) and indicative of longer, weaker bonds in the studied proteins. A positive linear correlation between (2h)JNN and the difference in imidazole ring (15)N chemical shift (Δ(15)N = |δ(15)Nδ1 - δ(15)Ne2|) was found to be consistent with variable H-bond length and variable cap population related to the ionization of histidine in the capping and noncapping states. The relative ease of (2h)JNN detection suggests that this parameter can become part of the standard arsenal for describing histidines in helix caps and other key structural and catalytic elements involving NH···N H-bonds. The combined nucleic acid and protein data extend the utility of (2h)JNN as a sensitive marker of local structural, dynamic, and thermodynamic properties in biomolecules.

THB1 is one of several group 1 truncated hemoglobins (TrHb1s) encoded in the genome of the unicellular green algaChlamydomonas reinhardtii. THB1 expression is under the control of NIT2, the master regulator of nitrate assimilation, which also controls the expression of the only nitrate reductase in the cell, NIT1.In vitroand physiological evidence suggests that THB1 converts the nitric oxide generated by NIT1 into nitrate. To aid in the elucidation of the function and mechanism of THB1, the structure of the protein was solved in the ferric state. THB1 resembles other TrHb1s, but also exhibits distinct features associated with the coordination of the heme iron by a histidine (proximal) and a lysine (distal). The new structure illustrates the versatility of the TrHb1 fold, suggests factors that stabilize the axial ligation of a lysine, and highlights the difficulty of predicting the identity of the distal ligand, if any, in this group of proteins.

THB1 is a ∼15 kDa “truncated” hemoglobin (Hb) found in the cytoplasm of the unicellular green alga Chlamydomonas reinhardtii. As for other Hbs within photosynthetic microbes, THB1 is thought to be involved in the management of reactive oxygen/nitrogen species. To complement physiological studies aimed at determining the function of THB1, we are pursuing the characterization of the recombinant protein (rTHB1) [1] with special attention to the heme environment. At pH ∼ 5, ferric rTHB1 has an optical spectrum characteristic of a six-coordinate, high-spin complex with a histidine and a water molecule as axial ligands to the heme iron. When the pH is raised near neutrality, the protein undergoes a transition (apparent pKa of 6.5) to a six-coordinate, low-spin complex. Mutagenesis and NMR data collected in the diamagnetic ferrous state provide direct evidence for the ligation of a lysine (K53) on the distal side of the heme [1]. Here, we continue the study of this unusual iron coordination scheme. The distal lysine, like the distal histidine of cyanobacterial globins (GlbNs), facilitates electron transfer. However, unlike the histidine of GlbN, the distal lysine of THB1 does not protect ferric heme from oxidative damage caused by H2O2. Analysis of ferrous THB1 NMR data reveals a neutral lysine amino group with highly upfield-shifted 15N signal. Fast-exchange averaging of the two amino protons is a further indication of a dynamic distal coordination. Additional structural information was obtained from diffraction data collected at beamline X25 (National Synchrotron Light Source, Brookhaven National laboratory). Crystals of ferric rTHB1 grown at pH 9.5 diffracted to 1.9 A resolution (P6122 space group) and the data were used to inspect the structural factors allowing lysine coordination.[1] Johnson et al. (2014) Biochemistry 53:4573Supported by NSF grant MCB-1330488

The hemoglobins of the cyanobacteria Synechococcus and Synechocystis (GlbNs) are capable of spontaneous and irreversible attachment of the b heme to the protein matrix. The reaction, which saturates the heme 2-vinyl by addition of a histidine residue, is reproduced in vitro by preparing the recombinant apoprotein, adding ferric heme, and reducing the iron to the ferrous state. Spontaneous covalent attachment of the heme is potentially useful for protein engineering purposes. Thus, to explore whether the histidine–heme linkage can serve in such applications, we attempted to introduce it in a test protein. We selected as our target the heme domain of Chlamydomonas eugametos LI637 (CtrHb), a eukaryotic globin that exhibits less than 50% sequence identity with the cyanobacterial GlbNs. We chose two positions, 75 in the FG corner and 111 in the H helix, to situate a histidine near a vinyl group. We characterized the proteins with gel electrophoresis, absorbance spectroscopy, and NMR analysis. Both T111H and L75H CtrHbs reacted upon reduction of the ferric starting material containing cyanide as the distal ligand to the iron. With L75H CtrHb, nearly complete (> 90%) crosslinking was observed to the 4-vinyl as expected from the X-ray structure of wild-type CtrHb. Reaction of T111H CtrHb also occurred at the 4-vinyl in a 60% yield, indicating a preference for the flipped heme orientation in the starting material. The work suggests that the His–heme modification will be applicable to the design of proteins with a non-dissociable heme group.

The nuclear genome of the model organism Chlamydomonas reinhardtii contains genes for a dozen hemoglobins of the truncated lineage. Of those, THB1 is known to be expressed, but the product and its function have not yet been characterized. We present mutagenesis, optical, and nuclear magnetic resonance data for the recombinant protein and show that at pH near neutral in the absence of added ligand, THB1 coordinates the heme iron with the canonical proximal histidine and a distal lysine. In the cyanomet state, THB1 is structurally similar to other known truncated hemoglobins, particularly the heme domain of Chlamydomonas eugametos LI637, a light-induced chloroplastic hemoglobin. Recombinant THB1 is capable of binding nitric oxide (NO(•)) in either the ferric or ferrous state and has efficient NO(•) dioxygenase activity. By using different C. reinhardtii strains and growth conditions, we demonstrate that the expression of THB1 is under the control of the NIT2 regulatory gene and that the hemoglobin is linked to the nitrogen assimilation pathway.

THB1 is one of twelve genes encoding hemoglobins in Chlamydomonas reinhardtii. Although previous work [1] has correlated the expression of THB1 to nitrate metabolism through the NIT2 gene regulator, the exact function of THB1 is unknown. Current work investigates possible enzymatic roles by examining the chemical and biophysical properties of recombinant THB1 (rTHB1) produced in E. coli as an apoprotein and recombined with a b heme. The goal of the present work was to validate the use of rTHB1 as a surrogate for the native protein. THB1 was partially purified from whole cell extracts of C. reinhardtii by chromatographic methods. Enhanced chemiluminescence staining and immunodetection of the protein mixture after native gel electrophoresis showed that THB1 has peroxidase activity. Ultra performance liquid chromatography and mass spectrometry confirmed the association of the polypeptide with a b heme and revealed that THB1 is N-terminally acetylated. Nanodrop analysis of the purified extracts returned an optical spectrum consistent with that of recombinant ferric THB1. In addition, the mixture had nitric oxide dioxygenase activity, as observed for rTHB1 [1]. The combined information provides compelling evidence that THB1 uses a b heme as cofactor and that the properties of rTHB1 are relevant to THB1 as it is found within the living cell. The observation of a co-translational modification cautions against the sole use of sequence information to derive physiological insight.[1] Johnson et al. (2014) Biochemistry 53:4573Supported by NSF grant MCB-1330488

The hemoglobin from the cyanobacterium Synechococcus sp. PCC 7002 (GlbN) contains three tyrosines (Tyr5, Tyr22, and Tyr53), each of which undergoes a structural rearrangement when the protein binds an exogenous ligand such as cyanide. We explored the use of 3-fluorotyrosine and (19)F-NMR spectroscopy for the characterization of GlbN. Assignment of (19)F resonances in fluorinated GlbN (GlbN*) was achieved with individual Tyr5Phe and Tyr53Phe replacements. We observed marked variations in chemical shift and linewidth reflecting the dependence of structural and dynamic properties on oxidation state, ligation state, and covalent attachment of the heme group. The isoelectronic complexes of ferric GlbN* with cyanide and ferrous GlbN* with carbon monoxide gave contrasting spectra, the latter exhibiting heterogeneity and enhanced internal motions on a microsecond-to-millisecond time scale. The strength of the H-bond network involving Tyr22 (B10) and bound cyanide was tested at high pH. 3-Fluorotyrosine at position 22 had a pK(a) value at least 3 units higher than its intrinsic value, 8.5. In addition, evidence was found for long-range communication among the tyrosine sites. These observations demonstrated the utility of the 3-fluorotyrosine approach to gain insight in hemoglobin properties.

GlbN, the “truncated” hemoglobin from Synechocystis sp. PCC 6803, coordinates the heme iron with His46 (E10, distal) and His70 (F8, proximal). Displacement of His E10 by an exogenous ligand (e.g., cyanide in the ferric state) drives a significant conformational change allowing Tyr22 (B10), Gln43 (E7) and Gln47 (E11) to establish a hydrogen bond network stabilizing the distal ligand.THB1, a closely related hemoglobin from Chlamydomonas reinhardtii, also binds cyanide and forms the same network of interactions. However, in the absence of an exogenous ligand, the neutral amino group of Lys53 (E10) coordinates the heme iron on the distal side [1]. Because of the importance of the coordination scheme in controlling the reactivity of heme proteins, the flexibility of the truncated hemoglobin fold, and the rarity of lysine coordination, we explored the ability of GlbN to use lysine at position E10 as an axial ligand. The His46Lys replacement yielded a protein with UV-Vis and NMR spectra similar to those of His46Leu and comparable pH response, suggesting that GlbN does not accommodate lysine coordination at position E10. However, the spectra were incompatible with water coordination and suggested that in the ferric state these variants were low-spin endogenously hexacoordinate complexes. We performed amino acid replacements within the distal H-bond network to characterize the perturbed heme environment and ligand sets. A combination of pH titrations and NMR experiments illustrates the delicate balance of interactions governing the heme pocket conformation of GlbN.[1] Johnson et al. (2014) Biochemistry 53:4573Supported by NSF grant MCB-1330488

Essentially all hemoglobins (Hbs) bind b heme reversibly, but notable exceptions are found in cyanobacterial 2/2 (“truncated”) Hbs. In the FeII state, the Hb from Synechocystis sp. PCC 6803 (GlbN) undergoes a posttranslational modification (PTM) in which a non-iron-coordinating histidine (H117) adds across the heme 2-vinyl. Heme PTMs alter the chemical properties of the prosthetic group and, as such, warrant investigation. The rarity of the histidine-heme crosslink in heme proteins adds interest to the PTM determinants. Here, we examined the robustness of the nucleophilic addition with amino acid replacements. We applied absorbance and NMR spectroscopies to characterize the products.We generated two GlbN variants. The first, H117A/L79H GlbN, was designed to eliminate PTM at the 2-vinyl and explore reactivity on the 4-vinyl side of the heme. Insertion of a histidine at position 79 caused a severe decrease in ferric heme affinity. Despite this, reduction to the FeII state resulted in a PTM identical to that observed with H117 in the wild-type protein. Remarkably, the second variant, L79H GlbN, exhibited reactivity at both the 2- and 4-heme vinyls, demonstrating a lack of stringent electronic requirements for the reaction.To test crosslink formation further, we used the 2/2 Hb from Chlamydomonaseugametos (Ce-Hb), which compared to GlbN has a similar fold, only 45% sequence identity, and distinct heme environment in the ligand-free state. We engineered histidine residues at the topological equivalents of H117 and L79 in GlbN (T111 and L75). With changes to the GlbN protocol involving the distal ligand, evidence for reductive heme modification was detected in both Ce Hb variants. The data suggested strongly that properly positioned histidines can cause heme PTM in hemoglobins.Supported by NSF MCB 0843439