A colorimetric method to measure in vitro nitrogenase functionality for engineering nitrogen fixation.

Biological nitrogen fixation (BNF) is the reduction of N2 into NH3 in a group of prokaryotes by an extremely O2-sensitive protein complex called nitrogenase. Transfer of the BNF pathway directly into plants, rather than by association with microorganisms, could generate crops that are less dependent on synthetic nitrogen fertilizers and increase agricultural productivity and sustainability. In the laboratory, nitrogenase activity is commonly determined by measuring ethylene produced from the nitrogenase-dependent reduction of acetylene (ARA) using a gas chromatograph. The ARA is not well suited for analysis of large sample sets nor easily adapted to automated robotic determination of nitrogenase activities. Here, we show that a reduced sulfonated viologen derivative (S2Vred) assay can replace the ARA for simultaneous analysis of isolated nitrogenase proteins using a microplate reader. We used the S2Vred to screen a library of NifH nitrogenase components targeted to mitochondria in yeast. Two NifH proteins presented properties of great interest for engineering of nitrogen fixation in plants, namely NifM independency, to reduce the number of genes to be transferred to the eukaryotic host; and O2 resistance, to expand the half-life of NifH iron-sulfur cluster in a eukaryotic cell. This study established that NifH from Dehalococcoides ethenogenes did not require NifM for solubility, [Fe-S] cluster occupancy or functionality, and that NifH from Geobacter sulfurreducens was more resistant to O2 exposure than the other NifH proteins tested. It demonstrates that nitrogenase components with specific biochemical properties such as a wider range of O2 tolerance exist in Nature, and that their identification should be an area of focus for the engineering of nitrogen-fixing crops.

Biosynthesis of cofactor-activatable iron-only nitrogenase in Saccharomyces cerevisiae.

Engineering nitrogenase in eukaryotes is hampered by its genetic complexity and by the oxygen sensitivity of its protein components. Of the three types of nitrogenases, the Fe-only nitrogenase is considered the simplest one because its function depends on fewer gene products than the homologous and more complex Mo and V nitrogenases. Here, we show the expression of stable Fe-only nitrogenase component proteins in the low-oxygen mitochondria matrix of S. cerevisiae. As-isolated Fe protein (AnfH) was active in electron donation to NifDK to reduce acetylene into ethylene. Ancillary proteins NifU, NifS and NifM were not required for Fe protein function. The FeFe protein existed as apo-AnfDK complex with the AnfG subunit either loosely bound or completely unable to interact with it. Apo-AnfDK could be activated for acetylene reduction by the simple addition of FeMo-co in vitro, indicating preexistence of the P-clusters even in the absence of coexpressed NifU and NifS. This work reinforces the use of Fe-only nitrogenase as simple model to engineer nitrogen fixation in yeast and plant mitochondria.

Exploiting genetic diversity and gene synthesis to identify superior nitrogenase NifH protein variants to engineer N2-fixation in plants.

Engineering nitrogen fixation in eukaryotes requires high expression of functional nitrogenase structural proteins, a goal that has not yet been achieved. Here we build a knowledge-based library containing 32 nitrogenase nifH sequences from prokaryotes of diverse ecological niches and metabolic features and combine with rapid screening in tobacco to identify superior NifH variants for plant mitochondria expression. Three NifH variants outperform in tobacco mitochondria and are further tested in yeast. Hydrogenobacter thermophilus (Aquificae) NifH is isolated in large quantities from yeast mitochondria and fulfills NifH protein requirements for efficient N2 fixation, including electron transfer for substrate reduction, P-cluster maturation, and FeMo-co biosynthesis. H. thermophilus NifH expressed in tobacco leaves shows lower nitrogenase activity than that from yeast. However, transfer of [Fe4S4] clusters from NifU to NifH in vitro increases 10-fold the activity of the tobacco-isolated NifH, revealing that plant mitochondria [Fe-S] cluster availability constitutes a bottleneck to engineer plant nitrogenases.

Use of synthetic biology tools to optimize the production of active nitrogenase Fe protein in chloroplasts of tobacco leaf cells.

The generation of nitrogen fixing crops is considered a challenge that could lead to a new agricultural 'green' revolution. Here, we report the use of synthetic biology tools to achieve and optimize the production of active nitrogenase Fe protein (NifH) in the chloroplasts of tobacco plants. Azotobacter vinelandii nitrogen fixation genes, nifH, M, U and S, were re-designed for protein accumulation in tobacco cells. Targeting to the chloroplast was optimized by screening and identifying minimal length transit peptides performing properly for each specific Nif protein. Putative peptidyl-prolyl cis-trans isomerase NifM proved necessary for NifH solubility in the stroma. Purified NifU, a protein involved in the biogenesis of NifH [4Fe-4S] cluster, was found functional in NifH reconstitution assays. Importantly, NifH purified from tobacco chloroplasts was active in the reduction of acetylene to ethylene, with the requirement of nifU and nifS co-expression. These results support the suitability of chloroplasts to host functional nitrogenase proteins, paving the way for future studies in the engineering of nitrogen fixation in higher plant plastids and describing an optimization pipeline that could also be used in other organisms and in the engineering of new metabolic pathways in plastids.

Purification and In Vitro Activity of Mitochondria Targeted Nitrogenase Cofactor Maturase NifB.

Active NifB is a milestone in the process of engineering nitrogen fixing plants. NifB is an extremely O2-sensitive S-adenosyl methionine (SAM)-radical enzyme that provides the key metal cluster intermediate (NifB-co) for the biosyntheses of the active-site cofactors of all three types of nitrogenases. NifB and NifB-co are unique to diazotrophic organisms. In this work, we have expressed synthetic codon-optimized versions of NifB from the γ-proteobacterium Azotobacter vinelandii and the thermophilic methanogen Methanocaldococcus infernus in Saccharomyces cerevisiae and in Nicotiana benthamiana. NifB proteins were targeted to the mitochondria, where O2 consumption is high and bacterial-like [Fe-S] cluster assembly operates. In yeast, NifB proteins were co-expressed with NifU, NifS, and FdxN proteins that are involved in NifB [Fe-S] cluster assembly and activity. The synthetic version of thermophilic NifB accumulated in soluble form within the yeast cell, while the A. vinelandii version appeared to form aggregates. Similarly, NifB from M. infernus was expressed at higher levels in leaves of Nicotiana benthamiana and accumulated as a soluble protein while A. vinelandii NifB was mainly associated with the non-soluble cell fraction. Soluble M. infernus NifB was purified from aerobically grown yeast and biochemically characterized. The purified protein was functional in the in vitro FeMo-co synthesis assay. This work presents the first active NifB protein purified from a eukaryotic cell, and highlights the importance of screening nif genes from different organisms in order to sort the best candidates to assemble a functional plant nitrogenase.

Formation of Nitrogenase NifDK Tetramers in the Mitochondria of Saccharomyces cerevisiae.

Transferring the prokaryotic enzyme nitrogenase into a eukaryotic host with the final aim of developing N2 fixing cereal crops would revolutionize agricultural systems worldwide. Targeting it to mitochondria has potential advantages because of the organelle's high O2 consumption and the presence of bacterial-type iron-sulfur cluster biosynthetic machinery. In this study, we constructed 96 strains of Saccharomyces cerevisiae in which transcriptional units comprising nine Azotobacter vinelandii nif genes (nifHDKUSMBEN) were integrated into the genome. Two combinatorial libraries of nif gene clusters were constructed: a library of mitochondrial leading sequences consisting of 24 clusters within four subsets of nif gene expression strength, and an expression library of 72 clusters with fixed mitochondrial leading sequences and nif expression levels assigned according to factorial design. In total, 29 promoters and 18 terminators were combined to adjust nif gene expression levels. Expression and mitochondrial targeting was confirmed at the protein level as immunoblot analysis showed that Nif proteins could be efficiently accumulated in mitochondria. NifDK tetramer formation, an essential step of nitrogenase assembly, was experimentally proven both in cell-free extracts and in purified NifDK preparations. This work represents a first step toward obtaining functional nitrogenase in the mitochondria of a eukaryotic cell.

Expression of a functional oxygen-labile nitrogenase component in the mitochondrial matrix of aerobically grown yeast.

The extreme sensitivity of nitrogenase towards oxygen stands as a major barrier to engineer biological nitrogen fixation into cereal crops by direct nif gene transfer. Here, we use yeast as a model of eukaryotic cell and show that aerobically grown cells express active nitrogenase Fe protein when the NifH polypeptide is targeted to the mitochondrial matrix together with the NifM maturase. Co-expression of NifH and NifM with Nif-specific Fe-S cluster biosynthetic proteins NifU and NifS is not required for Fe protein activity, demonstrating NifH ability to incorporate endogenous mitochondrial Fe-S clusters. In contrast, expression of active Fe protein in the cytosol requires both anoxic growth conditions and co-expression of NifH and NifM with NifU and NifS. Our results show the convenience of using mitochondria to host nitrogenase components, thus providing instrumental technology for the grand challenge of engineering N2-fixing cereals.

Identification of SUMO targets by a novel proteomic approach in plants(F).

Post-translational modifications (PTMs) chemically and physically alter the properties of proteins, including their folding, subcellular localization, stability, activity, and consequently their function. In spite of their relevance, studies on PTMs in plants are still limited. Small Ubiquitin-like Modifier (SUMO) modification regulates several biological processes by affecting protein-protein interactions, or changing the subcellular localizations of the target proteins. Here, we describe a novel proteomic approach to identify SUMO targets that combines 2-D liquid chromatography, immunodetection, and mass spectrometry (MS) analyses. We have applied this approach to identify nuclear SUMO targets in response to heat shock. Using a bacterial SUMOylation system, we validated that some of the targets identified here are, in fact, labeled with SUMO1. Interestingly, we found that GIGANTEA (GI), a photoperiodic-pathway protein, is modified with SUMO in response to heat shock both in vitro and in vivo.

A bacterial cysteine protease effector protein interferes with photosynthesis to suppress plant innate immune responses.

The bacterial pathogen Pseudomonas syringae pv tomato DC3000 suppresses plant innate immunity with effector proteins injected by a type III secretion system (T3SS). The cysteine protease effector HopN1, which reduces the ability of DC3000 to elicit programmed cell death in non-host tobacco, was found to also suppress the production of defence-associated reactive oxygen species (ROS) and callose when delivered by Pseudomonas fluorescens heterologously expressing a P. syringae T3SS. Purified His(6) -tagged HopN1 was used to identify tomato PsbQ, a member of the oxygen evolving complex of photosystem II (PSII), as an interacting protein. HopN1 localized to chloroplasts and both degraded PsbQ and inhibited PSII activity in chloroplast preparations, whereas a HopN1(D299A) non-catalytic mutant lost these abilities. Gene silencing of NtPsbQ in tobacco compromised ROS production and programmed cell death by DC3000. Our data reveal PsbQ as a contributor to plant immunity responses and a target for pathogen suppression.

Regulation of the new Arabidopsis imprinted gene AtBMI1C requires the interplay of different epigenetic mechanisms.

Recently, it has been shown that plants contain homologs to the animal Polycomb repressive complex 1 (PRC1) components BMI1 and RING1A/B. In Arabidopsis, there are three BMI1-like genes, two of which, AtBMI1A and B, are required during post-embryonic plant growth to repress embryonic traits and allow cell differentiation. However, little is known about the third BMI1-like gene, AtBMI1C. In this work, we show that AtBMI1C is only expressed during endosperm and stamen development. AtBMI1C is an imprinted gene expressed from the maternal allele in the endosperm but biallelically expressed in stamen. We found that the characteristic expression pattern of AtBMI1C is the result of a complex epigenetic regulation that involves CG DNA methylation, RNA-directed non-CG DNA methylation (RdDM), and PcG activity. Our results show the orchestrated interplay of different epigenetic mechanisms in regulating gene expression throughout development, shedding light on the current hypotheses for the origin and mechanism of imprinting in plant endosperm.

Identification of a protein network interacting with TdRF1, a wheat RING ubiquitin ligase with a protective role against cellular dehydration.

Plants exploit ubiquitination to modulate the proteome with the final aim to ensure environmental adaptation and developmental plasticity. Ubiquitination targets are specifically driven to degradation through the action of E3 ubiquitin ligases. Genetic analyses have indicated wide functions of ubiquitination in plant life; nevertheless, despite the large number of predicted E3s, only a few of them have been characterized so far, and only a few ubiquitination targets are known. In this work, we characterized durum wheat (Triticum durum) RING Finger1 (TdRF1) as a durum wheat nuclear ubiquitin ligase. Moreover, its barley (Hordeum vulgare) homolog was shown to protect cells from dehydration stress. A protein network interacting with TdRF1 has been defined. The transcription factor WHEAT BEL1-TYPE HOMEODOMAIN1 (WBLH1) was degraded in a TdRF1-dependent manner through the 26S proteasome in vivo, the mitogen-activated protein kinase TdWNK5 [for Triticum durum WITH NO LYSINE (K)5] was able to phosphorylate TdRF1 in vitro, and the RING-finger protein WHEAT VIVIPAROUS-INTERACTING PROTEIN2 (WVIP2) was shown to have a strong E3 ligase activity. The genes coding for the TdRF1 interactors were all responsive to cold and/or dehydration stress, and a negative regulative function in dehydration tolerance was observed for the barley homolog of WVIP2. A role in the control of plant development was previously known, or predictable based on homology, for wheat BEL1-type homeodomain1(WBLH1). Thus, TdRF1 E3 ligase might act regulating the response to abiotic stress and remodeling plant development in response to environmental constraints.

The Arabidopsis cell cycle F-box protein SKP2A binds to auxin.

Arabidopsis thaliana S-Phase Kinase-Associated Protein 2A (SKP2A) is an F-box protein that regulates the proteolysis of cell cycle transcription factors. The plant hormone auxin regulates multiple aspects of plant growth and development, including cell division. We found that auxin induces the ubiquitin-dependent degradation of SKP2A both in vivo and in vitro, suggesting that this hormone acts as a signal to trigger SKP2A proteolysis. In this article, we show that auxin binds directly and specifically to SKP2A. By TIR1-based superposition and docking analyzes, we identified an auxin binding site in SKP2A. Mutations in this binding site reduce the ability of SKP2A to bind to auxin and generate nondegradable SKP2A forms. In addition, these non-auxin binding proteins are unable to promote E2FC/DPB degradation in vivo or to induce cell division in the root meristem. Auxin binds to TIR1 to promote its interaction with the auxin/indole-3-acetic acid target proteins. Here, we show that auxin also enhanced the interaction between SKP2A and DPB. Finally, a mutation in SKP2A leads to auxin-resistant root growth, an effect that is additive with the tir1-1 phenotype. Thus, our data indicate that SKP2A is an auxin binding protein that connects auxin signaling with cell division.

Keeping cell identity in Arabidopsis requires PRC1 RING-finger homologs that catalyze H2A monoubiquitination.

Polycomb group (PcG) proteins form conserved regulatory complexes that modify chromatin to repress the genes that are not required in a specific differentiation status [1]. In animals, the two best-characterized PcG complexes are PRC2 and PRC1, which respectively possess histone 3 lysine 27 (H3K27) trimethyltransferase [2-4] and histone 2A lysine 119 (H2AK119) E3 ubiquitin ligase activities [5-7]. In Arabidopsis, PRC2 activity is also required for the gene silencing mechanism [8]; however, the existence of PRC1 has been questioned, because plant genomes do not encode clear PRC1 components and H2A monoubiquitination has not been detected [6, 9]. Conversely, recent reports have unveiled the presence of homologs to PRC1 components that together with plant-specific proteins could be part of the long-sought PRC1-like complexes [10, 11]. Here we show that the PRC1 RING-finger homologs AtBMI1A and AtBMI1B are implicated in the repression of embryonic and stem cell regulators. Plants impaired in AtBMI1A and AtBMI1B show derepression of embryonic traits in somatic cells, displaying a phenotype similar to plants mutant in PRC2 components [12-14]. Our data demonstrate that the AtBMI1A/B proteins mediate H2A monoubiquitination in Arabidopsis and that this mark, together with PRC2-mediated H3K27 trimethylation, plays a key role in maintaining cell identity.

A role for AUXIN RESISTANT3 in the coordination of leaf growth.

The characteristically flat structure of Arabidopsis thaliana vegetative leaves requires coordinating the growth of the epidermal, palisade mesophyll, spongy mesophyll and vascular tissues. Mutations disrupting such coordination or the specific growth properties of any of these tissues can cause hyponasty, epinasty, waviness or other deviations from flatness. Here, we show that the incurvata6 (icu6) semi-dominant allele of the AUXIN RESISTANT3 (AXR3) gene causes leaf hyponasty. Cotyledons and leaves of icu6/AXR3 plants exhibited reduced size of adaxial pavement cells, and abnormal expansion of palisade mesophyll cells. Enhanced auxin responses in the adaxial domain of icu6/AXR3 developing cotyledons and leaves correlated with increased cell divisions in the adaxial epidermis. Leaf incurvature in icu6/AXR3 leaves was alleviated by loss-of-function alleles of the ASYMMETRIC LEAVES1 (AS1) and AS2 genes, which restrict the expression of class I KNOX genes to the shoot apical meristem and regulate cell proliferation in leaf primordia. Taken together, our results suggest that an interaction between auxin responses and the AS1-AS2 pathway coordinates tissue growth during Arabidopsis thaliana leaf expansion.

Identification of ubiquitinated proteins in Arabidopsis.

Ubiquitin (Ub) is a small peptide that is covalently attached to proteins in a posttranslational reaction. Ubiquitination is a precise regulatory system that is present in all eukaryotic organisms and regulates the stability, the activity, the localization and the transport of proteins. Ubiquitination involves different enzymatic activities, in which the E3 ligases catalyze the last step recruiting of the target for labelling with ubiquitin. Genomic analyses have shown that the ubiquitin-proteasome system involves a large number of proteins in plants, as approximately 5% of the total protein belongs to this pathway. In contrast to the high number of E3 ligases of ubiquitin identified, very few proteins regulated by ubiquitination have been described. To solve this, we have undertaken a new proteomic approach aimed to identify proteins modified with ubiquitin. This is based on affinity purification and identification for ubiquitinated proteins using the ubiquitin binding domain (UBA) polypeptide of the P62 protein attached to agarose beads. This P62-agarose matrix is capable of specifically binding ubiquitinated proteins. These bound proteins were digested with trypsin and the peptides separated by HPLC chromatography, spotted directly onto a MALDI target and analyzed by MALDI-TOF/TOF off-line coupled LC/MALDI-MS/MS. A total of 200 putative ubiquitinated proteins were identified. From these we found that several of the putative targets were already described in plants, as well as in other organisms, as ubiquitinated proteins. In addition, we have found that some of these proteins were indeed modified with ubiquitin in vivo. Taken together, we have shown that this approach is useful for identifying ubiquitinated protein in plants.

Assessing allergen levels in peach and nectarine cultivars.

BACKGROUND: The lipid transfer protein Pru p 3 has been identified as a major peach fruit allergen. However, the putative peach member of the Bet v 1 family, Pru p 1, has been neither identified nor characterized. OBJECTIVES: To determine the distribution and solubility properties of the main peach allergens and to quantify Pru p 3 and Pru p 1 levels in peach and nectarine cultivars. METHODS: Peach peel and pulp were extracted using different buffers, and extracts were analyzed by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunodetection using polyclonal antibodies against lipid transfer proteins, profilins, and Bet v 1 homologues. Pru p 3 was quantified in peach and nectarine cultivars using a sandwich enzyme-linked immunosorbent assay method. A similar method was developed to quantify Pru p 1. RESULTS: A differential distribution between peel and pulp and different solubility properties were found for Pru p 3, Pru p 1, and peach profilin. Mean Pru p 3 levels were 132.86, 0.61, and 16.92 microg/g of fresh weight of peels, pulps, and whole fruits, respectively. The corresponding mean Pru p 1 levels were 0.62, 0.26, and 0.09 microg/g of fresh weight. Most US cultivars showed higher levels of both allergens than Spanish cultivars. CONCLUSIONS: The different distribution and solubility properties of the main peach allergens can determine the quality of fruit extracts used as diagnostic tools. These differences, together with the natural variation of Pru p 3 and Pru p 1 levels among peach and nectarine cultivars, can be exploited to reduce peach allergenicity by means of industrial processing and plant breeding.

An experimental and modeling-based approach to locate IgE epitopes of plant profilin allergens.

BACKGROUND: Plant profilins are actin-binding proteins that form a well-known panallergen family responsible for cross-sensitization between plant foods and pollens. Melon profilin, Cuc m 2, is the major allergen of this fruit. OBJECTIVE: We sought to map IgE epitopes on the 3-dimensional structure of Cuc m 2. METHODS: IgE binding to synthetic peptides spanning the full Cuc m 2 amino acid sequence was assayed by using a serum pool and individual sera from 10 patients with melon allergy with significant specific IgE levels to this allergen. Three-dimensional modeling and potential epitope location were based on analysis of both solvent exposure and electrostatic properties of the Cuc m 2 surface. RESULTS: Residues included in synthetic peptides that exerted the strongest IgE-binding capacity defined 2 major epitopes (E1, consisting of residues 66-75 and 81-93, and E2, consisting of residues 95-99 and 122-131) that partially overlapped with the actin-binding site of Cuc m 2. Two additional epitopes (E3, including residues 2-10, and E4, including residues 35-45) that should show weaker putative antigen-antibody associations and shared most residues with synthetic peptides with low IgE-binding capacity were predicted on theoretical grounds. CONCLUSIONS: Strong and weak IgE epitopes have been uncovered in melon profilin, Cuc m 2. CLINICAL IMPLICATIONS: The different types of IgE epitopes located in the 3-dimensional structure of melon profilin can constitute the molecular basis to explain the sensitization and cross-reactivity exhibited by this panallergen family.

Differential allergen sensitization patterns in chestnut allergy with or without associated latex-fruit syndrome.

BACKGROUND: Chestnut allergy has been almost exclusively considered in the context of the latex-fruit syndrome. Chestnut allergens not linked to latex hypersensitivity have not been studied. OBJECTIVE: We sought to explore whether differences in sensitization patterns between chestnut allergy with or without associated latex-fruit syndrome can be detected. METHODS: Twelve patients sensitized to chestnut but not to latex and 3 control patients with latex-chestnut allergy were analyzed. A major chestnut allergen was purified and characterized. IgE immunoblotting, specific IgE determination, and skin prick tests with 5 isolated allergens involved in food allergy or latex-fruit syndrome were also performed. RESULTS: A major 9-kd allergen was detected in chestnut extract, isolated, and identified as lipid transfer protein (LTP) Cas s 8. Specific IgE to this allergen was found in 91% (by means of IgE immunoblotting) and 58% (by means of ELISA) of sera from patients with chestnut but not latex allergy. Moreover, 66% of these patients had positive skin prick test responses to Cas s 8. Additionally, allergenic LTPs from peach fruit and Artemisia vulgaris pollen were also reactive. In contrast, avocado class I chitinase and latex hevein, allergens associated with the latex-fruit syndrome, showed no reaction. The opposite situation was exhibited by patients with latex-chestnut allergy. CONCLUSIONS: Patients with chestnut allergy with or without associated latex hypersensitivity present different patterns of major allergens (LTPs and class I chitinases, respectively). CLINICAL IMPLICATIONS: LTPs and class I chitinases can be used as diagnostic tools in patients with chestnut allergy to predict whether an associated latex sensitization and a risk of potential cross-reactivity with other plant foods and pollens exist.