Polyamines Involved in Regulating Self-Incompatibility in Apple

Apple exhibits typical gametophytic self-incompatibility, in which self-S-RNase can arrest pollen tube growth, leading to failure of fertilization. To date, there have been few studies on how to resist the toxicity of self-S-RNase. In this study, pollen tube polyamines were found to respond to self-S-RNase and help pollen tubes defend against self-S-RNase. In particular, the contents of putrescine, spermidine, and spermine in the pollen tube treated with self-S-RNase were substantially lower than those treated with non-self-S-RNase. Further analysis of gene expression of key enzymes in the synthesis and degradation pathways of polyamines found that the expression of DIAMINE OXIDASE 4 (MdDAO4) as well as several polyamine oxidases such as POLYAMINE OXIDASES 3 (MdPAO3), POLYAMINE OXIDASES 4 (MdPAO4), and POLYAMINE OXIDASES 6 (MdPAO6) were significantly up-regulated under self-S-RNase treatment, resulting in the reduction of polyamines. Silencing MdPAO6 in pollen tubes alleviates the inhibitory effect of self-S-RNase on pollen tube growth. In addition, exogenous polyamines also enhance pollen tube resistance to self-S-RNase. Transcriptome sequencing data found that polyamines may communicate with S-RNase through the calcium signal pathway, thereby regulating the growth of the pollen tubes. To summarize, our results suggested that polyamines responded to the self-incompatibility reaction and could enhance pollen tube tolerance to S-RNase, thus providing a potential way to break self-incompatibility in apple.

The tree of life of polyamine oxidases

Polyamine oxidases (PAOs) are characterized by a broad variability in catalytic properties and subcellular localization, and impact key cellular processes in diverse organisms. In the present study, a comprehensive phylogenetic analysis was performed to understand the evolution of PAOs across the three domains of life and particularly within eukaryotes. Phylogenetic trees show that PAO-like sequences of bacteria, archaea, and eukaryotes form three distinct clades, with the exception of a few procaryotes that probably acquired a PAO gene through horizontal transfer from a eukaryotic donor. Results strongly support a common origin for archaeal PAO-like proteins and eukaryotic PAOs, as well as a shared origin between PAOs and monoamine oxidases. Within eukaryotes, four main lineages were identified that likely originated from an ancestral eukaryotic PAO before the split of the main superphyla, followed by specific gene losses in each superphylum. Plant PAOs show the highest diversity within eukaryotes and belong to three distinct clades that underwent to multiple events of gene duplication and gene loss. Peptide deletion along the evolution of plant PAOs of Clade I accounted for further diversification of function and subcellular localization. This study provides a reference for future structure–function studies and emphasizes the importance of extending comparisons among PAO subfamilies across multiple eukaryotic superphyla.

Polyamine Oxidases Play Various Roles in Plant Development and Abiotic Stress Tolerance

Polyamines not only play roles in plant growth and development, but also adapt to environmental stresses. Polyamines can be oxidized by copper-containing diamine oxidases (CuAOs) and flavin-containing polyamine oxidases (PAOs). Two types of PAOs exist in the plant kingdom; one type catalyzes the back conversion (BC-type) pathway and the other catalyzes the terminal catabolism (TC-type) pathway. The catabolic features and biological functions of plant PAOs have been investigated in various plants in the past years. In this review, we focus on the advance of PAO studies in rice, Arabidopsis, and tomato, and other plant species.

A Polyamine Oxidase from Selaginella lepidophylla (SelPAO5) can Replace AtPAO5 in Arabidopsis through Converting Thermospermine to Norspermidine instead to Spermidine

Of the five polyamine oxidases in Arabidopsis thaliana, AtPAO5 has a substrate preference for the tetraamine thermospermine (T-Spm) which is converted to triamine spermidine (Spd) in a back-conversion reaction in vitro. A homologue of AtPAO5 from the lycophyte Selaginella lepidophylla (SelPAO5) back-converts T-Spm to the uncommon polyamine norspermidine (NorSpd) instead of Spd. An Atpao5 loss-of-function mutant shows a strong reduced growth phenotype when growing on a T-Spm containing medium. When SelPAO5 was expressed in the Atpao5 mutant, T-Spm level decreased to almost normal values of wild type plants, and NorSpd was produced. Furthermore the reduced growth phenotype was cured by the expression of SelPAO5. Thus, a NorSpd synthesis pathway by PAO reaction and T-Spm as substrate was demonstrated in planta and the assumption that a balanced T-Spm homeostasis is needed for normal growth was strengthened.

Engineering enzyme catalysis: an inverse approach

Enzymes’ inherent chirality confers their exquisite enantiomeric specificity and makes their use as green alternatives to chiral metal complexes or chiral organocatalysts invaluable to the fine chemical industry. The most prevalent way to alter enzyme activity in terms of regioselectivity and stereoselectivity for both industry and fundamental research is to engineer the enzyme. In a recent article by Keinänen et al., published in Bioscience Reports 2018, ‘Controlling the regioselectivity and stereoselectivity of FAD-dependent polyamine oxidases with the use of amine-attached guide molecules as conformational modulators’, an inverse approach was presented that focuses on the manipulation of the enzyme substrate rather than the enzyme. This approach not only uncovered dormant enantioselectivity in related enzymes but allowed for its control by the use of guide molecules simply added to the reaction solution or covalently linked to an achiral scaffold molecule.

Controlling the regioselectivity and stereospecificity of FAD-dependent polyamine oxidases with the use of amine-attached guide molecules as conformational modulators

Enzymes generally display strict stereospecificity and regioselectivity for their substrates. Here by using FAD-dependent human acetylpolyamine oxidase (APAO), human spermine (Spm) oxidase (SMOX) and yeast polyamine oxidase (Fms1), we demonstrate that these fundamental properties of the enzymes may be regulated using simple guide molecules, being either covalently attached to polyamines or used as a supplement to the substrate mixtures. APAO, which naturally metabolizes achiral N1-acetylated polyamines, displays aldehyde-controllable stereospecificity with chiral 1-methylated polyamines, like (R)- and (S)-1-methylspermidine (1,8-diamino-5-azanonane) (1-MeSpd). Among the novel N1-acyl derivatives of MeSpd, isonicotinic acid (P4) or benzoic acid (Bz) with (R)-MeSpd had Km of 3.6 ± 0.6/1.2 ± 0.7 µM and kcat of 5.2 ± 0.6/4.6 ± 0.7 s−1 respectively, while N1-AcSpd had Km 8.2 ± 0.4 µM and kcat 2.7 ± 0.0 s−1. On the contrary, corresponding (S)-MeSpd amides were practically inactive (kcat < 0.03 s−1) but they retained micromole level Km for APAO. SMOX did not metabolize any of the tested compounds (kcat < 0.05 s−1) that acted as non-competitive inhibitors having Ki ≥ 155 µM for SMOX. In addition, we tested (R,R)-1,12-bis-methylspermine (2,13-diamino-5,10-diazatetradecane) (R,R)-(Me2Spm) and (S,S)-Me2Spm as substrates for Fms1. Fms1 preferred (S,S)- to (R,R)-diastereoisomer, but with notably lower kcat in comparison with spermine. Interestingly, Fms1 was prone to aldehyde supplementation in its regioselectivity, i.e. the cleavage site of spermidine. Thus, aldehyde supplementation to generate aldimines or N-terminal substituents in polyamines, i.e. attachment of guide molecule, generates novel ligands with altered charge distribution changing the binding and catalytic properties with polyamine oxidases. This provides means for exploiting hidden capabilities of polyamine oxidases for controlling their regioselectivity and stereospecificity.

Identification and expression profiling of flax (Linum usitatissimum L.) polyamine oxidase genes in response to stimuli

Polyamine oxidases (PAOs) are known to be involved in either the terminal catabolism or the back conversion of polyamines, which affect a range of physiological processes, including growth, development, and stress responses. In this study, based on genome-wide analysis, we identified five putative PAO genes (LuPAO1toLuPAO5) in flax (Linum usitatissimumL.) that contain the amino-oxidase domain and FAD-binding-domain. The expression analysis using quantitative real-time PCR revealed spatial variations in the expression ofLuPAOsin different organs. In addition, the expression level ofLuPAOsin the flax cell suspension culture was increased by treatment with methyl-jasmonate (MeJA) or pectin, but not with salicylic acid or chitosan. This indicates that LuPAOs might be involved in the MeJA-mediated biological activities. Taken together, our genome-wide analysis of PAO genes and expression profiling of these genes provide the first step toward the functional dissection of LuPAOs.