2',3'-cAMP treatment mimics the stress molecular response in Arabidopsis thaliana.

The role of the RNA degradation product 2',3'-cyclic adenosine monophosphate (2',3'-cAMP) is poorly understood. Recent studies have identified 2',3'-cAMP in plant material and determined its role in stress signaling. The level of 2',3'-cAMP increases upon wounding, in the dark, and under heat, and 2',3'-cAMP binding to an RNA-binding protein, Rbp47b, promotes stress granule (SG) assembly. To gain further mechanistic insights into the function of 2',3'-cAMP, we used a multi-omics approach by combining transcriptomics, metabolomics, and proteomics to dissect the response of Arabidopsis (Arabidopsis thaliana) to 2',3'-cAMP treatment. We demonstrated that 2',3'-cAMP is metabolized into adenosine, suggesting that the well-known cyclic nucleotide-adenosine pathway of human cells might also exist in plants. Transcriptomics analysis revealed only minor overlap between 2',3'-cAMP- and adenosine-treated plants, suggesting that these molecules act through independent mechanisms. Treatment with 2',3'-cAMP changed the levels of hundreds of transcripts, proteins, and metabolites, many previously associated with plant stress responses, including protein and RNA degradation products, glucosinolates, chaperones, and SG components. Finally, we demonstrated that 2',3'-cAMP treatment influences the movement of processing bodies, confirming the role of 2',3'-cAMP in the formation and motility of membraneless organelles.

Structural Determinants of Sugar Alcohol Biosynthesis in Plants: The Crystal Structures of Mannose-6-Phosphate and Aldose-6-Phosphate Reductases.

Sugar alcohols are major photosynthetic products in plant species from the Apiaceae and Plantaginaceae families. Mannose-6-phosphate reductase (Man6PRase) and aldose-6-phosphate reductase (Ald6PRase) are key enzymes for synthesizing mannitol and glucitol in celery (Apium graveolens) and peach (Prunus persica), respectively. In this work, we report the first crystal structures of dimeric plant aldo/keto reductases (AKRs), celery Man6PRase (solved in the presence of mannonic acid and NADP+) and peach Ald6PRase (obtained in the apo form). Both structures displayed the typical TIM barrel folding commonly observed in proteins from the AKR superfamily. Analysis of the Man6PRase holo form showed that residues putatively involved in the catalytic mechanism are located close to the nicotinamide ring of NADP+, where the hydride transfer to the sugar phosphate should take place. Additionally, we found that Lys48 is important for the binding of the sugar phosphate. Interestingly, the Man6PRase K48A mutant had a lower catalytic efficiency with mannose-6-phosphate but a higher catalytic efficiency with mannose than the wild type. Overall, our work sheds light on the structure-function relationships of important enzymes to synthesize sugar alcohols in plants.

Cofactor Specificity Switch on Peach Glucitol Dehydrogenase.

Most oxidoreductases that use NAD+ or NADP+ to transfer electrons in redox reactions display a strong preference for the cofactor. The catalytic efficiency of peach glucitol dehydrogenase (GolDHase) for NAD+ is 1800-fold higher than that for NADP+. Herein, we combined structural and kinetic data to reverse the cofactor specificity of this enzyme. Using site-saturation mutagenesis, we obtained the D216A mutant, which uses both NAD+ and NADP+, although with different catalytic efficiencies (1000 ± 200 and 170 ± 30 M-1 s-1, respectively). This mutant was used as a template to introduce further mutations by site-directed mutagenesis, using information from the fruit fly NADP-dependent GolDHase. The D216A/V217R/D218S triple mutant displayed a 2-fold higher catalytic efficiency with NADP+ than with NAD+. Overall, our results indicate that the triple mutant has the potential to be used for metabolic and cellular engineering and for cofactor recycling in industrial processes.

Carbohydrate Metabolism in Bacteria: Alternative Specificities in ADP-Glucose Pyrophosphorylases Open Novel Metabolic Scenarios and Biotechnological Tools.

We explored the ability of ADP-glucose pyrophosphorylase (ADP-Glc PPase) from different bacteria to use glucosamine (GlcN) metabolites as a substrate or allosteric effectors. The enzyme from the actinobacteria Kocuria rhizophila exhibited marked and distinctive sensitivity to allosteric activation by GlcN-6P when producing ADP-Glc from glucose-1-phosphate (Glc-1P) and ATP. This behavior is also seen in the enzyme from Rhodococcus spp., the only one known so far to portray this activation. GlcN-6P had a more modest effect on the enzyme from other Actinobacteria (Streptomyces coelicolor), Firmicutes (Ruminococcus albus), and Proteobacteria (Agrobacterium tumefaciens) groups. In addition, we studied the catalytic capacity of ADP-Glc PPases from the different sources using GlcN-1P as a substrate when assayed in the presence of their respective allosteric activators. In all cases, the catalytic efficiency of Glc-1P was 1-2 orders of magnitude higher than GlcN-1P, except for the unregulated heterotetrameric protein (GlgC/GgD) from Geobacillus stearothermophilus. The Glc-1P substrate preference is explained using a model of ADP-Glc PPase from A. tumefaciens based on the crystallographic structure of the enzyme from potato tuber. The substrate-binding domain localizes near the N-terminal of an α-helix, which has a partial positive charge, thus favoring the interaction with a hydroxyl rather than a charged primary amine group. Results support the scenario where the ability of ADP-Glc PPases to use GlcN-1P as an alternative occurred during evolution despite the enzyme being selected to use Glc-1P and ATP for α-glucans synthesis. As an associated consequence in such a process, certain bacteria could have improved their ability to metabolize GlcN. The work also provides insights in designing molecular tools for producing oligo and polysaccharides with amino moieties.

Proteogenic Dipeptides Are Characterized by Diel Fluctuations and Target of Rapamycin Complex-Signaling Dependency in the Model Plant Arabidopsis thaliana.

As autotrophic organisms, plants capture light energy to convert carbon dioxide into ATP, nicotinamide adenine dinucleotide phosphate (NADPH), and sugars, which are essential for the biosynthesis of building blocks, storage, and growth. At night, metabolism and growth can be sustained by mobilizing carbon (C) reserves. In response to changing environmental conditions, such as light-dark cycles, the small-molecule regulation of enzymatic activities is critical for reprogramming cellular metabolism. We have recently demonstrated that proteogenic dipeptides, protein degradation products, act as metabolic switches at the interface of proteostasis and central metabolism in both plants and yeast. Dipeptides accumulate in response to the environmental changes and act via direct binding and regulation of critical enzymatic activities, enabling C flux distribution. Here, we provide evidence pointing to the involvement of dipeptides in the metabolic rewiring characteristics for the day-night cycle in plants. Specifically, we measured the abundance of 13 amino acids and 179 dipeptides over short- (SD) and long-day (LD) diel cycles, each with different light intensities. Of the measured dipeptides, 38 and eight were characterized by day-night oscillation in SD and LD, respectively, reaching maximum accumulation at the end of the day and then gradually falling in the night. Not only the number of dipeptides, but also the amplitude of the oscillation was higher in SD compared with LD conditions. Notably, rhythmic dipeptides were enriched in the glucogenic amino acids that can be converted into glucose. Considering the known role of Target of Rapamycin (TOR) signaling in regulating both autophagy and metabolism, we subsequently investigated whether diurnal fluctuations of dipeptides levels are dependent on the TOR Complex (TORC). The Raptor1b mutant (raptor1b), known for the substantial reduction of TOR kinase activity, was characterized by the augmented accumulation of dipeptides, which is especially pronounced under LD conditions. We were particularly intrigued by the group of 16 dipeptides, which, based on their oscillation under SD conditions and accumulation in raptor1b, can be associated with limited C availability or photoperiod. By mining existing protein-metabolite interaction data, we delineated putative protein interactors for a representative dipeptide Pro-Gln. The obtained list included enzymes of C and amino acid metabolism, which are also linked to the TORC-mediated metabolic network. Based on the obtained results, we speculate that the diurnal accumulation of dipeptides contributes to its metabolic adaptation in response to changes in C availability. We hypothesize that dipeptides would act as alternative respiratory substrates and by directly modulating the activity of the focal enzymes.

Biochemical characterization of recombinant UDP-sugar pyrophosphorylase and galactinol synthase from Brachypodium distachyon.

Raffinose (Raf) protects plant cells during seed desiccation and under different abiotic stress conditions. The biosynthesis of Raf starts with the production of UDP-galactose by UDP-sugar pyrophosphorylase (USPPase) and continues with the synthesis of galactinol by galactinol synthase (GolSase). Galactinol is then used by Raf synthase to produce Raf. In this work, we report the biochemical characterization of USPPase (BdiUSPPase) and GolSase 1 (BdiGolSase1) from Brachypodium distachyon. The catalytic efficiency of BdiUSPPase was similar with galactose 1-phosphate and glucose 1-phosphate, but 5- to 17-fold lower with other sugar 1-phosphates. The catalytic efficiency of BdiGolSase1 with UDP-galactose was three orders of magnitude higher than with UDP-glucose. A structural model of BdiGolSase1 allowed us to determine the residues putatively involved in the binding of substrates. Among these, we found that Cys261 lies within the putative catalytic pocket. BdiGolSase1 was inactivated by oxidation with diamide and H2O2. The activity of the diamide-oxidized enzyme was recovered by reduction with dithiothreitol or E. coli thioredoxin, suggesting that BdiGolSase1 is redox-regulated.