Adaptive diversity in structure and function of C4 photosynthetic components.

Many tropical and subtropical plant lineages have independently evolved C4 photosynthesis. The convergent evolution of this complex functional trait from different ancestors is reflected in variations in the structural and biochemical characteristics of C4 components such as enzymes and cellular specializations. The mechanism of C4 carbon concentration mostly involves coordinated function of mesophyll and bundle sheath cells. Important adaptations of the C4 syndrome include increased vein density and the development of photosynthetic bundle sheath cells with low gas conductance. In addition, the enzymes and transporters of the C4 pathway evolved via the co-option of multiple genes, each derived from a specific lineage of isoforms present in nonC4-ancestors. In particular, the adaptation of C4 enzymes resulted in a variety of structural and biochemical modifications, generally leading to increased catalytic efficiency and regulation by metabolites and post-translational modifications. Differences in these adaptations are particularly evident in the C4-acid decarboxylation step, which can be catalyzed by three decarboxylases that define the C4 subtypes. Associated with the biochemical subtypes, there are also differences in the extend of grana staking and localization of bundle sheath cells chloroplasts. The presence of a suberin layer and symplastic connections also likely vary among the different C4-subtypes. This review examines the current understanding of the diversity of structural and functional changes in key components of the C4 carbon concentration mechanism. This knowledge is necessary not only to identify divergent solutions for convergent optimization of C4 components in different C4 lineages, but also to guide their creation for rational synthetic biology approaches.

A122S, A205V, D376E, W574L and S653N substitutions in acetolactate synthase (ALS) from Amaranthus palmeri show different functional impacts on herbicide resistance.

BACKGROUND: Amaranthus palmeri S. Watson, a problematic weed infesting summer crops in Argentina, has developed multiple herbicide resistance. Resistance to acetolactate synthase (ALS)-inhibiting herbicides is particularly common, with high-level resistance mostly caused by different mutations in the ALS enzyme. Six versions of the enzyme were identified from a resistant A. palmeri population, carrying substitutions D376E, A205V, A122S, A282D, W574L and S653N. This work aims to provide a comparative analysis of these mutants and the wild-type (WT) enzyme to fully understand the herbicide resistance. Thus, all the versions of the ALS gene from A. palmeri were heterologously expressed and purified to evaluate their kinetics and inhibitory response against imazethapyr, diclosulam, chlorimuron-ethyl, flucarbazone-sodium and bispyribac-sodium. RESULTS: A decrease in catalytic efficiency was detected in the A205V, A122S-A282D, W574L and S653N ApALS enzymes, whereas only A205V and W574L substitutions also produced a decrease in the substrate affinity. In vitro ALS inhibition assays confirmed cross-resistance to almost all the herbicides tested, with the exception of A282D ApALS, which was as susceptible as WT ApALS. Moreover, the results confirmed that the novel substitution A122S provides cross-resistance to at least one herbicide within each of the five families of ALS inhibitors, and this property could be explained by a lower number of hydrophobic interactions between the herbicides and the mutant enzyme. CONCLUSION: This is the first report to compare various mutations in vitro from A. palmeri ALS. Our data contribute to understanding the impacts of herbicide resistance in this species. © 2021 Society of Chemical Industry.

BlsA Is a Low to Moderate Temperature Blue Light Photoreceptor in the Human Pathogen Acinetobacter baumannii.

Light is an environmental signal that produces extensive effects on the physiology of the human pathogen Acinetobacter baumannii. Many of the bacterial responses to light depend on BlsA, a bluelight using FAD (BLUF)-type photoreceptor, which also integrates temperature signals. In this work, we disclose novel mechanistic aspects of the function of BlsA. First, we show that light modulation of motility occurs only at temperatures lower than 24°C, a phenotype depending on BlsA. Second, blsA transcript levels were significantly reduced at temperatures higher than 25°C, in agreement with BlsA protein levels in the cell which were undetectable at 26°C and higher temperatures. Also, quantum yield of photo-activation of BlsA (lBlsA) between 14 and 37°C, showed that BlsA photoactivity is greatly compromised at 25°C and absent above 28°C. Fluorescence emission and anisotropy of the cofactor together with the intrinsic protein fluorescence studies suggest that the FAD binding site is more susceptible to structural changes caused by increments in temperature than other regions of the protein. Moreover, BlsA itself gains structural instability and strongly aggregates at temperatures above 30°C. Overall, BlsA is a low to moderate temperature photoreceptor, whose functioning is highly regulated in the cell, with control points at expression of the cognate gene as well as photoactivity.

Posttranslational Modification of the NADP-Malic Enzyme Involved in C4 Photosynthesis Modulates the Enzymatic Activity during the Day.

Evolution of the C4 photosynthetic pathway involved in some cases recruitment of housekeeping proteins through gene duplication and their further neofunctionalization. NADP-malic enzyme (ME), the most widespread C4 decarboxylase, has increased its catalytic efficiency and acquired regulatory properties that allowed it to participate in the C4 pathway. Here, we show that regulation of maize (Zea mays) C4-NADP-ME activity is much more elaborate than previously thought. Using mass spectrometry, we identified phosphorylation of the Ser419 residue of C4-NADP-ME in protein extracts of maize leaves. The phosphorylation event increases in the light, with a peak at Zeitgeber time 2. Phosphorylation of ZmC4-NADP-ME drastically decreases its activity as shown by the low residual activity of the recombinant phosphomimetic mutant. Analysis of the crystal structure of C4-NADP-ME indicated that Ser419 is involved in the binding of NADP at the active site. Molecular dynamics simulations and effective binding energy computations indicate a less favorable binding of the cofactor NADP in the phosphomimetic and the phosphorylated variants. We propose that phosphorylation of ZmC4-NADP-ME at Ser419 during the first hours in the light is a cellular mechanism that fine tunes the enzymatic activity to coordinate the carbon concentration mechanism with the CO2 fixation rate, probably to avoid CO2 leakiness from bundle sheath cells.

Molecular adaptations of NADP-malic enzyme for its function in C4 photosynthesis in grasses.

In C4 grasses of agronomical interest, malate shuttled into the bundle sheath cells is decarboxylated mainly by nicotinamide adenine dinucleotide phosphate (NADP)-malic enzyme (C4-NADP-ME). The activity of C4-NADP-ME was optimized by natural selection to efficiently deliver CO2 to Rubisco. During its evolution from a plastidic non-photosynthetic NADP-ME, C4-NADP-ME acquired increased catalytic efficiency, tetrameric structure and pH-dependent inhibition by its substrate malate. Here, we identified specific amino acids important for these C4 adaptions based on strict differential conservation of amino acids, combined with solving the crystal structures of maize and sorghum C4-NADP-ME. Site-directed mutagenesis and structural analyses show that Q503, L544 and E339 are involved in catalytic efficiency; E339 confers pH-dependent regulation by malate, F140 is critical for the stabilization of the oligomeric structure and the N-terminal region is involved in tetramerization. Together, the identified molecular adaptations form the basis for the efficient catalysis and regulation of one of the central biochemical steps in C4 metabolism.

Deciphering the number and location of active sites in the monomeric glyoxalase I of Zea mays.

Detoxification of methylglyoxal, a toxic by-product of central sugar metabolism, is a major issue for all forms of life. The glyoxalase pathway evolved to effectively convert methylglyoxal into d-lactate via a glutathione hemithioacetal intermediate. Recently, we have shown that the monomeric glyoxalase I from maize exhibits a symmetric fold with two cavities, potentially harboring two active sites, in analogy with homodimeric enzyme surrogates. Here we confirm that only one of the two cavities exhibits glyoxalase I activity and show that it adopts a tunnel-shaped structure upon substrate binding. Such conformational change gives rise to independent binding sites for glutathione and methylglyoxal in the same active site, with important implications for the molecular reaction mechanism, which has been a matter of debate for several decades. DATABASE: Structural data are available in The Protein Data Bank database under the accession numbers 6BNN, 6BNX, and 6BNZ.

A novel triple amino acid substitution in the EPSPS found in a high-level glyphosate-resistant Amaranthus hybridus population from Argentina.

BACKGROUND: The evolution of herbicide-resistant weeds is one of the most important concerns of global agriculture. Amaranthus hybridus L. is a competitive weed for summer crops in South America. In this article, we intend to unravel the molecular mechanisms by which an A. hybridus population from Argentina has become resistant to extraordinarily high levels of glyphosate. RESULTS: The glyphosate-resistant population (A) exhibited particularly high parameters of resistance (GR50 = 20 900 g ai ha-1 , Rf = 314), with all plants completing a normal life cycle even after 32X dose application. No shikimic acid accumulation was detected in the resistant plants at any of the glyphosate concentrations tested. Molecular and genetic analyses revealed a novel triple substitution (TAP-IVS: T102I, A103V, and P106S) in the 5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS) enzyme of population A and an incipient increase on the epsps relative copy number but without effects on the epsps transcription levels. The novel mechanism was prevalent, with 48% and 52% of the individuals being homozygous and heterozygous for the triple substitution, respectively. In silico conformational studies revealed that TAP-IVS triple substitution would generate an EPSPS with a functional active site but with an increased restriction to glyphosate binding. CONCLUSION: The prevalence of the TAP-IVS triple substitution as the sole mechanism detected in the highly glyphosate resistant population suggests the evolution of a new glyphosate resistance mechanism arising in A. hybridus. This is the first report of a naturally occurring EPSPS triple substitution and the first glyphosate target-site resistance mechanism described in A. hybridus. © 2018 Society of Chemical Industry.

Metabolic regulation of phytoplasma malic enzyme and phosphotransacetylase supports the use of malate as an energy source in these plant pathogens.

Phytoplasmas ('Candidatus Phytoplasma') are insect-vectored plant pathogens. The genomes of these bacteria are small with limited metabolic capacities making them dependent on their plant and insect hosts for survival. In contrast to mycoplasmas and other relatives in the class Mollicutes, phytoplasmas encode genes for malate transporters and malic enzyme (ME) for conversion of malate into pyruvate. It was hypothesized that malate is probably a major energy source for phytoplasmas as these bacteria are limited in the uptake and processing of carbohydrates. In this study, we investigated the metabolic capabilities of 'Candidatus (Ca.) phytoplasma' aster yellows witches'-broom (AYWB) malic enzyme (ME). We found that AYWB-ME has malate oxidative decarboxylation activity, being able to convert malate to pyruvate and CO2 with the reduction of either NAD or NADP, and displays distinctive kinetic mechanisms depending on the relative concentration of the substrates. AYWB-ME activity was strictly modulated by the ATP/ADP ratio, a feature which has not been found in other ME isoforms characterized to date. In addition, we found that the 'Ca. Phytoplasma' AYWB PduL-like enzyme (AYWB-PduL) harbours phosphotransacetylase activity, being able to convert acetyl-CoA to acetyl phosphate downstream of pyruvate. ATP also inhibited AYWB-PduL activity, as with AYWB-ME, and the product of the reaction catalysed by AYWB-PduL, acetyl phosphate, stimulated AYWB-ME activity. Overall, our data indicate that AYWB-ME and AYWB-PduL activities are finely coordinated by common metabolic signals, like ATP/ADP ratios and acetyl phosphate, which support their participation in energy (ATP) and reducing power [NAD(P)H] generation from malate in phytoplasmas.

Circadian oscillation and development-dependent expression of glycine-rich RNA binding proteins in tomato fruits.

Glycine-rich RNA-binding proteins (GRPs) are involved in the modulation of the post-transcriptional processing of transcripts and participate as an output signal of the circadian clock. However, neither GRPs nor the circadian rhythmic have been studied in detail in fleshy fruits as yet. In the present work, the GRP1 gene family was analysed in Micro-Tom tomato (Solanum lycopersicum L.) fruit. Three highly homologous LeGRP1 genes (LeGRP1a-c) were identified. For each gene, three products were found, corresponding to the unspliced precursor mRNA (pre-mRNA), the mature mRNA and the alternatively spliced mRNA (preLeGRP1a-c, mLeGRP1a-c and asLeGRP1a-c, respectively). Tomato GRPs (LeGRPs) show the classic RNA recognition motif and glycine-rich region, and were found in the nucleus and in the cytosol of tomato fruit. By using different Escherichia coli mutants, it was found that LeGRP1s contained in vivo RNA-melting abilities and were able to complement the cold-sensitive phenotype of BX04 cells. Particular circadian profiles of expression, dependent on the fruits' developmental stage, were found for each LeGRP1 form. During ripening off the vine of fruits harvested at the mature green stage, the levels of all LeGRP1a-c forms drastically increased; however, incubation at 4°C prevented such increases. Analysis of the expression of all LeGRP1a-c forms suggests a positive regulation of expression in tomato fruit. Overall, the results obtained in this work reveal a complex pattern of expression of GRPs in tomato fruit, suggesting they might be involved in post-transcriptional modulation of circadian processes of this fleshy fruit.

Biochemical approaches to C4 photosynthesis evolution studies: the case of malic enzymes decarboxylases.

C4 photosynthesis enables the capture of atmospheric CO2 and its concentration at the site of RuBisCO, thus counteracting the negative effects of low atmospheric levels of CO2 and high atmospheric levels of O2 (21 %) on photosynthesis. The evolution of this complex syndrome was a multistep process. It did not occur by simply recruiting pre-exiting components of the pathway from C3 ancestors which were already optimized for C4 function. Rather it involved modifications in the kinetics and regulatory properties of pre-existing isoforms of non-photosynthetic enzymes in C3 plants. Thus, biochemical studies aimed at elucidating the functional adaptations of these enzymes are central to the development of an integrative view of the C4 mechanism. In the present review, the most important biochemical approaches that we currently use to understand the evolution of the C4 isoforms of malic enzyme are summarized. It is expected that this information will help in the rational design of the best decarboxylation processes to provide CO2 for RuBisCO in engineering C3 species to perform C4 photosynthesis.

Kinetics and functional diversity among the five members of the NADP-malic enzyme family from Zea mays, a C4 species.

NADP-malic enzyme (NADP-ME) is involved in different metabolic pathways in several organisms due to the relevant physiological functions of the substrates and products of its reaction. In plants, it is one of the most important proteins that were recruited to fulfil key roles in C4 photosynthesis. Recent advances in genomics allowed the characterization of the complete set of NADP-ME genes from some C3 species, as Arabidopsis thaliana and Oryza sativa; however, the characterization of the complete NADP-ME family from a C4 species has not been performed yet. In this study, while taking advantage of the complete Zea mays genome sequence recently released, the characterization of the whole NADP-ME family is presented. The maize NADP-ME family is composed of five genes, two encoding plastidic NADP-MEs (ZmC4- and ZmnonC4-NADP-ME), and three cytosolic enzymes (Zmcyt1-, Zmcyt2-, and Zmcyt3-NADP-ME). The results presented clearly show that each maize NADP-ME displays particular organ distribution, response to stress stimuli, and differential biochemical properties. Phylogenetic footprinting studies performed with the NADP-MEs from several grasses, indicate that four members of the maize NADP-ME family share conserved transcription factor binding motifs with their orthologs, indicating conserved physiological functions for these genes in monocots. Based on the results obtained in this study, and considering the biochemical plasticity shown by the NADP-ME, it is discussed the relevance of the presence of a multigene family, in which each member encodes an isoform with particular biochemical properties, in the evolution of the C4 NADP-ME, improved to fulfil the requirements for an efficient C4 mechanism.

Plastidial NADP-malic enzymes from grasses: unraveling the way to the C4 specific isoforms.

Malic enzyme is present in many plant cell compartments such as plastids, cytosol and mitochondria. Particularly relevant is the plastidial isoform that participates in the C(4) cycle providing CO(2) to RuBisCO in C(4) species. This type of photosynthesis is more frequent among grasses where anatomical preconditioning would have facilitated the evolution of the C(4) syndrome. In maize (C(4) grass), the photosynthetic NADP dependent Malic enzyme (ZmC(4)-NADP-ME, l-malate:NADP oxidoreductase, E.C. 1.1.1.40) and the closest related non-photosynthetic isoform (ZmnonC(4)-NADP-ME, l-malate:NADP oxidoreductase, E.C. 1.1.1.40) are both plastidial but differ in expression pattern, kinetics and structure. Features like high catalytic efficiency, inhibition by high malate concentration at pH 7.0, redox modulation and tetramerization are characteristic of the photosynthetic NADP-ME. In this work, the proteins encoded by sorghum (C(4) grass) and rice (C(3) grass) NADP-ME genes, orthologues of the plastidial NADP-MEs from maize, were recombinantly expressed, purified and characterized. In a global comparison, we could identify a small group of residues which may explain the special features of C(4) enzymes. Overall, the present work presents biochemical and molecular data that helps to elucidate the changes that took place in the evolution of C(4) NADP-ME in grasses.

Functional characterization of residues involved in redox modulation of maize photosynthetic NADP-malic enzyme activity.

Two highly similar plastidic NADP-malic enzymes (NADP-MEs) are found in the C(4) species maize (Zea mays); one exclusively expressed in the bundle sheath cells (BSCs) and involved in C(4) photosynthesis (ZmC(4)-NADP-ME); and the other (ZmnonC(4)-NADP-ME) with housekeeping roles. In the present work, these two NADP-MEs were analyzed regarding their redox-dependent activity modulation. The results clearly show that ZmC(4)-NADP-ME is the only one modulated by redox status, and that its oxidation produces a conformational change limiting the catalytic process, although inducing higher affinity binding of the substrates. The reversal of ZmC(4)-NADP-ME oxidation by chemical reductants suggests the presence of thiol groups able to form disulfide bonds. In order to identify the cysteine residues involved in the activity modulation, site-directed mutagenesis and MALDI-TOF (matrix-assisted laser desorption ionization-time of flight) analysis of ZmC(4)-NADP-ME were performed. The results obtained allowed the identification of Cys192, Cys246 (not conserved in ZmnonC(4)-NADP-ME), Cys270 and Cys410 as directly or indirectly implicated in ZmC(4)-NADP-ME redox modulation. These residues may be involved in forming disulfide bridge(s) or in the modulation of the oxidation of critical residues. Overall, the results indicate that, besides having acquired a high level of expression and localization in BSCs, ZmC(4)-NADP-ME displays a particular redox modulation, which may be required to accomplish the C(4) photosynthetic metabolism. Therefore, the present work could provide new insights into the regulatory mechanisms potentially involved in the recruitment of genes for the C(4) pathway during evolution.

Maize cytosolic NADP-malic enzyme (ZmCytNADP-ME): a phylogenetically distant isoform specifically expressed in embryo and emerging roots.

Two maize plastidic NADP-malic enzyme isoforms have been characterized: the bundle sheath-located photosynthetic isoform (ZmC(4)-NADP-ME) and a constitutively expressed one (Zm-nonC(4)-NADP-ME). In this work, the characterization of the first maize cytosolic NADP-ME (ZmCytNADP-ME) is presented, which transcript is exclusively found in embryo and emerging roots. ZmCytNADP-ME expression in roots decreases with development, while Zm-nonC ( 4 ) -NADP-ME increases concomitantly. On the other hand, ZmCytNADP-ME accumulation is differentially modulated by several stress conditions and shows coordination with that of Zm-nonC ( 4 ) -NADP-ME in maize young roots. Recombinant ZmCytNADP-ME displays clearly distinct kinetic parameters and metabolic regulation than the plastidic isoforms. The particular properties and the specific-expression pattern of this novel isoform suggest that it may be involved in the control of cytosolic malate levels in emerging roots, e.g. during hypoxia. ZmCytNADP-ME is phylogenetically related to other cytosolic mono and dicot NADP-MEs, and data indicate that it belongs to an ancestral unique group among plant NADP-MEs.

Identification of domains involved in tetramerization and malate inhibition of maize C4-NADP-malic enzyme.

C(4) photosynthetic NADP-malic enzyme (ME) has evolved from non-C(4) isoforms and gained unique kinetic and structural properties during this process. To identify the domains responsible for the structural and kinetic differences between maize C(4) and non-C(4)-NADP-ME several chimeras between these isoforms were constructed and analyzed. By using this approach, we found that the region flanked by amino acid residues 102 and 247 is critical for the tetrameric state of C(4)-NADP-ME. In this way, the oligomerization strategy of these NADP-ME isoforms differs markedly from the one that present non-plant NADP-ME with known crystal structures. On the other hand, the region from residue 248 to the C-terminal end of the C(4) isoform is involved in the inhibition by high malate concentrations at pH 7.0. The inhibition pattern of the C(4)-NADP-ME and some of the chimeras suggested an allosteric site responsible for such behavior. This pH-dependent inhibition could be important for regulation of the C(4) isoform in vivo, with the enzyme presenting maximum activity while photosynthesis is in progress.