Pathogenesis-related (PR) proteins play important roles in plant defense response. However, functional investigation of PR10 genes is still limited and their physiological roles have not been conclusively characterized in biological processes of conifer trees. Here, we identified multiple novel members in the western white pine (Pinus monticola) PmPR10 family by bioinformatic mining available transcriptomic data. Phylogenetic analysis of protein sequences revealed four PR10 and two PR10-like clusters with a high synteny across different species of five-needle pines. Of 10 PmPR10 genes, PmPR10-3.1 was selected and expressed in Escherichia coli. The purified recombinant protein exhibited inhibitory effects on spore hyphal growth of fungal pathogens Cronartium ribicola, Phoma exigua, and Phoma argillacea by in-vitro anti-fungal analysis. Genetic variation analysis detected a total of 21 single nucleotide polymorphisms (SNPs) within PmPR10-3.1 in a collection of P. monticola seed families. A nonsynonymous SNP (t178g) showed significant association with relative levels of quantitative disease resistance (QDR), explaining about 8.7% of phenotypic variation as the peak value across all SNPs. Our results provide valuable insight into the genetic architecture underlying P. monticola QDR and imply that PmPR10-3.1 may function as an important component in conifer basal immunity for non-specific resistance to a wide spectrum of pathogens.

The reactions of protein oxidation play a significant role in many biological processes, especially in diseases development. Therefore, it is important to understand, how the protein molecule behaves in the presence of oxidants. In the present work, photo-oxidation of phytohormone-binding plant protein (VrPhBP) was investigated using light and 3-carboxybenzophenone (3CB) as a sensitizer (one electron oxidant). The protein interacts with the sensitizer in the ground state forming a weak binding complex leading to the presence of bound and free 3CB in solution. The early events and transient species (such as radicals and radical ions) formed during irradiation were characterised by transient spectroscopy showing the formation of the sulphur radical cation Met>S●+ (stabilized by (S∴N)+)and the tyrosyl radical TyrO● on VrPhBP. Thus the 3CB excited triplet state was quenched by the Met and Tyr residues and mostly by Met (based on the deconvoluted transient absorption spectra).The presence of a Tyr side chain in the vicinity of a Met residue results in intramolecular electron transfer from Tyr to the Met>S●+ radical cation, leading to regeneration of the thioether side chain and formation of TyrO●. The presence of other side chains close to Met, such as Arg or Lys can induce the stabilization of Met>S●+ via the formation of two-centered three-electron bonded species (S∴N)+. The transient species were additionally confirmed by stable product analysis. Based on SDS-PAGE, chromatography and mass spectrometry, the formation of methionine sulphoxide and Met-3CB adduct was identified together with di-Tyr cross links. On the basis of the experimental results the overall mechanism of VrPhBP photo-oxidation, from its early events to the formation of stable products, is described. In addition, a good correlation between the mechanisms of photooxidation of model compounds such as Met derivatives and peptides and those for real biological systems is emphasized.

Inorganic pyrophosphatases (PPases, EC 3.6.1.1), which hydrolyze inorganic pyrophosphate to phosphate in the presence of divalent metal cations, play a key role in maintaining phosphorus homeostasis in cells. DNA coding inorganic pyrophosphatases from Arabidopsis thaliana ( At PPA1) and Medicago truncatula ( Mt PPA1) were cloned into a bacterial expression vector and the proteins were produced in Escherichia coli cells and crystallized. In terms of their subunit fold, At PPA1 and Mt PPA1 are reminiscent of other members of Family I soluble pyrophosphatases from bacteria and yeast. Like their bacterial orthologs, both plant PPases form hexamers, as confirmed in solution by multi-angle light scattering and size-exclusion chromatography. This is in contrast to the fungal counterparts, which are dimeric. Unexpectedly, the crystallized At PPA1 and Mt PPA1 proteins lack ~30 amino acid residues at their N-termini, as independently confirmed by chemical sequencing. In vitro , self-cleavage of the recombinant proteins is observed after prolonged storage or during crystallization. The cleaved fragment corresponds to a putative signal peptide of mitochondrial targeting, with a predicted cleavage site at Val31-Ala32. Site-directed mutagenesis shows that mutations of the key active site Asp residues dramatically reduce the cleavage rate, which suggests a moonlighting proteolytic activity. Moreover, the discovery of autoproteolytic (rather than mitochondrion-mediated) cleavage of a mitochondrial-targeting peptide would change our perception of this signaling process.

Ll PR ‐10.2B, a Pathogenesis‐related class 10 ( PR ‐10) protein from yellow lupine ( Lupinus luteus ) was crystallized in complex with melatonin, an emerging important plant regulator and antioxidant. The structure reveals two molecules of melatonin bound in the internal cavity of the protein, plus a very well‐defined electron density near the cavity entrance, corresponding to an unknown ligand molecule comprised of two flat rings, which is most likely a product of melatonin transformation. In a separate Ll PR ‐10.2B co‐crystallization experiment with an equimolar mixture of melatonin and trans ‐zeatin, which is a cytokinin phytohormone well recognized as a PR ‐10‐binding partner, a quaternary 1 : 1 : 1 : 1 complex was formed, in which one of the melatonin‐binding sites has been substituted with trans ‐zeatin, whereas the binding of melatonin at the second binding site and binding of the unknown ligand are undisturbed. This unusual complex, when compared with the previously described PR ‐10/ trans ‐zeatin complexes and with the emerging structural information about melatonin binding by PR ‐10 proteins, provides intriguing insights into the role of PR ‐10 proteins in phytohormone regulation in plants, especially with the involvement of melatonin, and implicates the PR ‐10 proteins as low‐affinity melatonin binders under the conditions of elevated melatonin concentration. Databases Atomic coordinates and processed structure factors corresponding to the final models of the Ll PR ‐10.2B/melatonin and Ll PR ‐10.2B/melatonin + trans ‐zeatin complexes have been deposited with the Protein Data Bank ( PDB ) under the accession codes 5MXB and 5MXW . The corresponding raw X‐ray diffraction images have been deposited in the Rep OD Repository at the Interdisciplinary Centre for Mathematical and Computational Modelling ( ICM ) of the University of Warsaw, Poland, and are available for download with the following Digital Object Identifiers ( DOI ): https://doi.org/10.18150/repod.9923638 and https://doi.org/10.18150/repod.6621013 .

Plant pathogenesis-related class 10 (PR-10) proteins are a family of abundant proteins initially identified as elements of the plant defense system. The key structural feature suggesting PR-10 functionality is a huge hydrophobic cavity created in the protein interior by a scaffold composed of an extended β-sheet wrapped around a long and flexible C-terminal α-helix. Several crystallographic and NMR studies have shown that the cavity can accommodate a variety of small molecule ligands, including phytohormones. The article describes ∼1.3 Å resolution crystal structures of a Lupinus luteus PR-10 isoform LlPR-10.1A, in its free form and in complex with trans-zeatin, a naturally occurring plant hormone belonging to the cytokinin group. Moreover we present the structure of the same protein where the saturation with zeatin is not complete. This set of three crystal structures allows us to track the structural adaptation of the protein upon trans-zeatin docking, as well as the sequence of the ligand-binding events, step-by-step. In addition, titration of LlPR-10.1A with trans-zeatin monitored in solution by CD spectra, confirmed the pattern of structural adaptations deduced from the crystallographic studies. The ligand-biding mode shows no similarity to other zeatin complexes of PR-10 proteins. The present work, which describes the first atomic models of the same PR-10 protein with and without a physiological ligand, reveals that the conformation of LlPR-10.1A undergoes a significant structural rearrangement upon trans-zeatin binding.

Pathogenesis-related proteins of class 10 (PR-10) are a family of plant proteins with the same fold characterized by a large hydrophobic cavity that allows them to bind various ligands, such as phytohormones. A subfamily with only ∼20% sequence identity but with a conserved canonical PR-10 fold have previously been recognized as Cytokinin-Specific Binding Proteins (CSBPs), although structurally the binding mode oftrans-zeatin (a cytokinin phytohormone) was found to be quite diversified. Here, it is shown that two CSBP orthologues fromMedicago truncatulaandVigna radiatabind gibberellic acid (GA3), which is an entirely different phytohormone, in a conserved and highly specific manner. In both cases a single GA3 molecule is found in the internal cavity of the protein. The structural data derived from high-resolution crystal structures are corroborated by isothermal titration calorimetry (ITC), which reveals a much stronger interaction with GA3 than withtrans-zeatin and pH dependence of the binding profile. As a conclusion, it is postulated that the CSBP subfamily of plant PR-10 proteins should be more properly linked with general phytohormone-binding properties and termed phytohormone-binding proteins (PhBP).

ENWEndNote BIBJabRef, Mendeley RISPapers, Reference Manager, RefWorks, Zotero AMA Grzechowiak M, Sikorski M, Jaskolski M. Inorganic pyrophosphatase (PPase) from a higher plant. BioTechnologia. 2013;94(1):35-37. doi:10.5114/bta.2013.46433. APA Grzechowiak, M., Sikorski, M., & Jaskolski, M. (2013). Inorganic pyrophosphatase (PPase) from a higher plant. BioTechnologia, 94(1), 35-37. https://doi.org/10.5114/bta.2013.46433 Chicago Grzechowiak, Marta, Michał Sikorski, and Mariusz Jaskolski. 2013. "Inorganic pyrophosphatase (PPase) from a higher plant". BioTechnologia 94 (1): 35-37. doi:10.5114/bta.2013.46433. Harvard Grzechowiak, M., Sikorski, M., and Jaskolski, M. (2013). Inorganic pyrophosphatase (PPase) from a higher plant. BioTechnologia, 94(1), pp.35-37. https://doi.org/10.5114/bta.2013.46433 MLA Grzechowiak, Marta et al. "Inorganic pyrophosphatase (PPase) from a higher plant." BioTechnologia, vol. 94, no. 1, 2013, pp. 35-37. doi:10.5114/bta.2013.46433. Vancouver Grzechowiak M, Sikorski M, Jaskolski M. Inorganic pyrophosphatase (PPase) from a higher plant. BioTechnologia. 2013;94(1):35-37. doi:10.5114/bta.2013.46433.

DNA microarrays, which are among the most popular genomic tools, are widely applied in biology and medicine. Boutique arrays, which are small, spotted, dedicated microarrays, constitute an inexpensive alternative to whole-genome screening methods. The data extracted from each microarray-based experiment must be transformed and processed prior to further analysis to eliminate any technical bias. The normalization of the data is the most crucial step of microarray data pre-processing and this process must be carefully considered as it has a profound effect on the results of the analysis. Several normalization algorithms have been developed and implemented in data analysis software packages. However, most of these methods were designed for whole-genome analysis. In this study, we tested 13 normalization strategies (ten for double-channel data and three for single-channel data) available on R Bioconductor and compared their effectiveness in the normalization of four boutique array datasets. The results revealed that boutique arrays can be successfully normalized using standard methods, but not every method is suitable for each dataset. We also suggest a universal seven-step workflow that can be applied for the selection of the optimal normalization procedure for any boutique array dataset. The described workflow enables the evaluation of the investigated normalization methods based on the bias and variance values for the control probes, a differential expression analysis and a receiver operating characteristic curve analysis. The analysis of each component results in a separate ranking of the normalization methods. A combination of the ranks obtained from all the normalization procedures facilitates the selection of the most appropriate normalization method for the studied dataset and determines which methods can be used interchangeably.

Physical, chemical and biological stress factors, such as microbial infection, upregulate the transcription levels of a number of plant genes, coding for the so‐called pathogenesis‐related (PR) proteins. For PR proteins of class‐10 (PR‐10), the biological function remains unclear, despite two decades of scientific research. PR‐10 proteins have a wide distribution throughout the plant kingdom and the class members share size and secondary structure organization. Throughout the years, we and other groups have determined the structures of a number of PR‐10 proteins, both in the crystalline state by X‐ray diffraction and in solution by NMR spectroscopy. Despite the accumulating structural information, our understanding of PR‐10 function is still limited. PR‐10 proteins are rather small (~ 160 amino acids) with a fold consisting of three α helices and seven antiparallel β strands. These structural elements enclose a large hydrophobic cavity that is most probably the key to their functional relevance. Also, the outer surface of these proteins is of extreme interest, as epitopes from a PR‐10 subclass cause allergic reactions in humans.

Histidine‐containing phosphotransfer proteins (HPts) take part in hormone signal transduction in higher plants. The overall pathway of this process is reminiscent of the two‐component system initially identified in prokaryotes. HPts function in histidine–aspartate phosphorelays in which they mediate the signal from sensory kinases (usually membrane proteins) to RRs in the nucleus. Here, we report the crystal structure of an HPt protein from Medicago truncatula (MtHPt1) determined at 1.45 Å resolution and refined to an R‐factor of 16.7% using low‐temperature synchrotron‐radiation X‐ray diffraction data. There is one MtHPt1 molecule in the asymmetric unit of the crystal lattice with P212121 symmetry. The protein fold consists of six α helices, four of which form a C‐terminal helix bundle. The coiled‐coil structure of the bundle is stabilized by a network of S–aromatic interactions involving highly conserved sulfur‐containing residues. The structure reveals a solvent‐exposed side chain of His79, which is the phosphorylation site, as demonstrated by autoradiography combined with site‐directed mutation. It is surrounded by highly conserved residues present in all plant HPts. These residues form a putative docking interface for either the receiver domain of the sensory kinase, or for the RR. The biological activity of MtHPt1 was tested by autoradiography. It demonstrated phosphorylation by the intracellular kinase domain of the cytokinin receptor MtCRE1. Complex formation between MtHPt1 and the intracellular fragment of MtCRE1 was confirmed by thermophoresis, with a dissociation constant Kd of 14 μm.DatabaseThe atomic coordinates and structure factors for the crystal structure of histidine‐containing phosphotransfer protein MtHPt1 from Medicago truncatula have been deposited with the RCSB Protein Data Bank under the accession code 3us6.

ENWEndNote BIBJabRef, Mendeley RISPapers, Reference Manager, RefWorks, Zotero AMA Ruszkowski M, Sikorski M, Jaskolski M. Crystal structure of a PR-10 nodulin in complex with trans-zeatin. BioTechnologia. 2013;94(1):42-46. doi:10.5114/bta.2013.46436. APA Ruszkowski, M., Sikorski, M., & Jaskolski, M. (2013). Crystal structure of a PR-10 nodulin in complex with trans-zeatin. BioTechnologia, 94(1), 42-46. https://doi.org/10.5114/bta.2013.46436 Chicago Ruszkowski, Milosz, Michal Sikorski, and Mariusz Jaskolski. 2013. "Crystal structure of a PR-10 nodulin in complex with trans-zeatin". BioTechnologia 94 (1): 42-46. doi:10.5114/bta.2013.46436. Harvard Ruszkowski, M., Sikorski, M., and Jaskolski, M. (2013). Crystal structure of a PR-10 nodulin in complex with trans-zeatin. BioTechnologia, 94(1), pp.42-46. https://doi.org/10.5114/bta.2013.46436 MLA Ruszkowski, Milosz et al. "Crystal structure of a PR-10 nodulin in complex with trans-zeatin." BioTechnologia, vol. 94, no. 1, 2013, pp. 42-46. doi:10.5114/bta.2013.46436. Vancouver Ruszkowski M, Sikorski M, Jaskolski M. Crystal structure of a PR-10 nodulin in complex with trans-zeatin. BioTechnologia. 2013;94(1):42-46. doi:10.5114/bta.2013.46436.

ENWEndNote BIBJabRef, Mendeley RISPapers, Reference Manager, RefWorks, Zotero AMA Ciesielska A, Ruszkowski M, Kasperska A, Femiak I, Michalski Z, Sikorski M. REVIEW PAPERNew insights into the signaling and function of cytokinins in higher plants. BioTechnologia. 2012;93(4):400-413. doi:10.5114/bta.2012.46594. APA Ciesielska, A., Ruszkowski, M., Kasperska, A., Femiak, I., Michalski, Z., & Sikorski, M. (2012). REVIEW PAPERNew insights into the signaling and function of cytokinins in higher plants. BioTechnologia, 93(4), 400-413. https://doi.org/10.5114/bta.2012.46594 Chicago Ciesielska, Agnieszka, Milosz Ruszkowski, Alina Kasperska, Iwona Femiak, Zbigniew Michalski, and Michał M. Sikorski. 2012. "REVIEW PAPERNew insights into the signaling and function of cytokinins in higher plants". BioTechnologia 93 (4): 400-413. doi:10.5114/bta.2012.46594. Harvard Ciesielska, A., Ruszkowski, M., Kasperska, A., Femiak, I., Michalski, Z., and Sikorski, M. (2012). REVIEW PAPERNew insights into the signaling and function of cytokinins in higher plants. BioTechnologia, 93(4), pp.400-413. https://doi.org/10.5114/bta.2012.46594 MLA Ciesielska, Agnieszka et al. "REVIEW PAPERNew insights into the signaling and function of cytokinins in higher plants." BioTechnologia, vol. 93, no. 4, 2012, pp. 400-413. doi:10.5114/bta.2012.46594. Vancouver Ciesielska A, Ruszkowski M, Kasperska A, Femiak I, Michalski Z, Sikorski M. REVIEW PAPERNew insights into the signaling and function of cytokinins in higher plants. BioTechnologia. 2012;93(4):400-413. doi:10.5114/bta.2012.46594.

NodS is an S-adenosyl-L-methionine (SAM)-dependent N-methyltransferase that is involved in the biosynthesis of Nod factor (NF) in rhizobia, which are bacterial symbionts of legume plants. NF is a modified chitooligosaccharide (COS) signal molecule that is recognized by the legume host, where it initiates symbiotic processes leading to atmospheric nitrogen fixation. We report the crystal structure of recombinant NodS protein from Bradyrhizobium japonicum, which infects lupine and serradella legumes. Two crystal forms--ligand-free NodS and NodS in complex with S-adenosyl-L-homocysteine, which is a by-product of the methylation reaction--were obtained, and their structures were refined to resolutions of 2.43 Å and 1.85 Å, respectively. Although the overall fold (consisting of a seven-stranded β-sheet flanked by layers of helices) is similar to those of other SAM-dependent methyltransferases, NodS has specific features reflecting the unique character of its oligosaccharide substrate. In particular, the N-terminal helix and its connecting loop get ordered upon SAM binding, thereby closing the methyl donor cavity and shaping a long surface canyon that is clearly the binding site for the acceptor molecule. Comparison of the two structural forms of NodS suggests that there are also other conformational changes taking place upon the binding of the donor substrate. As an enzyme that methylates a COS substrate, NodS is the first example among all SAM-dependent methyltransferases to have its three-dimensional structure elucidated. Gaining insight about how NodS binds its donor and acceptor substrates helps to better understand the mechanism of NodS activity and the basis of its functional difference in various rhizobia.

Plant pathogenesis-related (PR) proteins of class 10 are the only group among the 17 PR protein families that are intracellular and cytosolic. Sequence conservation and the wide distribution of PR-10 proteins throughout the plant kingdom are an indication of an indispensable function in plants, but their true biological role remains obscure. Crystal and solution structures for several homologues have shown a similar overall fold with a vast internal cavity which, together with structural similarities to the steroidogenic acute regulatory protein-related lipid transfer domain and cytokinin-specific binding proteins, strongly indicate a ligand-binding role for the PR-10 proteins. This article describes the structure of a complex between a classic PR-10 protein [Lupinus luteus (yellow lupine) PR-10 protein of subclass 2, LlPR-10.2B] and N, N'-diphenylurea, a synthetic cytokinin. Synthetic cytokinins have been shown in various bioassays to exhibit activity similar to that of natural cytokinins. The present 1.95 A resolution crystallographic model reveals four N, N'-diphenylurea molecules in the hydrophobic cavity of the protein and a degree of conformational changes accompanying ligand binding. The structural adaptability of LlPR-10.2B and its ability to bind different cytokinins suggest that this protein, and perhaps other PR-10 proteins as well, can act as a reservoir of cytokinin molecules in the aqueous environment of a plant cell.

Hypericin, a red-colored naphtodianthrone, is a natural product synthesized in the medicinal plant Hypericum perforatum, widely known as St. John’s wort. Hypericin has been attracting a growing attention of the pharmaceutical industry because of its potential application in various therapies, including the treatment of depression. In vivo, hypericin is synthesized by dimerization of emodin in a complicated multistep reaction that is reportedly catalyzed by a small (17.8kDa) protein, Hyp-1. Based on relatively low sequence similarity (∼50%), Hyp-1 has been tentatively classified as a plant PR-10 (pathogenesis-related class 10) protein. Members of the PR-10 family are ubiquitous plant proteins associated with stress control and tissue differentiation but with no clearly understood molecular mechanism. They have, however, a well-defined folding canon, consisting of an extended antiparallel β-sheet wrapped around a C-terminal α-helix, enclosing in the protein interior a huge cavity, in which various hydrophobic ligands can be bound. Apart from Hyp-1, only two other PR-10 members have been found to possess enzymatic activity (S-norcoclaurine synthase and TcmN aromatase/cyclase). In this paper, we report a high-resolution crystal structure of Hyp-1, confirming that it indeed has a PR-10 fold. The protein binds multiple polyethylene glycol molecules, some of which occupy the hydrophobic cavity. The crystallographic model illustrates a high degree of conformational adaptability of both interacting partners for efficient binding. We have been unable, however, to dimerize emodin to hypericin using Hyp-1 as biocatalyst. This puzzling result does not have a clear explanation at this time.