Biology subjects

Filepova, R.

Publications and source records attributed to Filepova, R..

4 recordsLinked to original sources

bioRxiv2023

Study of auxin metabolism using stable isotope labeling and LCMS; evidence for in planta auxin decarboxylation pathway.

The natural plant hormone auxin indole-3-acetic acid (IAA) influences many physiological processes in plants. Here, the metabolism of IAA was studied in detail using tobacco BY-2 cells as a model and compared with the in planta metabolism in several plant species. A combination of labeled/unlabeled substrate feeding, global untargeted mass spectrometric (MS) scanning, and selective MS filtering allowed the detection of 17 auxin metabolites, 15 of which were identified. Subsequent study of intermediate metabolism and dynamics revealed eight major pathways: three amino acid conjugation pathways with aspartate, glutamate, and glutamine, followed by their 2-oxidation with the help of the DAO enzyme; side-chain glucosyl ester formation; direct 2-oxidation; two decarboxylation pathways; and a pathway producing an unidentified metabolite. Interestingly, the first intermediates of the two decarboxylation pathways, indole-3-carbinol and oxoindole-3-carbinol, were formed outside the cells. We found that the majority of the detected auxin metabolites occur naturally in several plant species and that IAA is their precursor, indicating that the auxin metabolic pathways observed in BY-2 cells also occur in planta. Our finding that the IAA decarboxylation pathway occurs in planta, and the previous reports of auxin activity of some metabolites of this pathway, suggest that at least some of the biological effects of IAA may be explained by its conversion to decarboxylative metabolites.

plant biology↗

bioRxiv2023

Phytohormone profiling in an evolutionary framework

Multiple phytohormones act as conserved developmental regulators in land plants. Although the closely related streptophyte green algae typically lack full complements of molecular pathways underlying these responses, scattered reports of endogenous phytohormone production in these organisms exist. In this study, we performed a detailed LC/MS-based analysis of several phytohormones, their precursors and metabolites in all lineages of streptophyte algae. We also included chlorophyte algae and early-diverging land plants as outgroups. Free auxin, tRNA-derived cytokinins and certain phenolics including salicylic acid were found ubiquitously. However, land plants differed from green algae by the consistent detection of abscisic acid and the presence of auxin and cytokinin conjugates and trans-zeatin, supporting the hypotheses that these three phytohormones likely came to regulate development in the ancestral land plant. By contrast, we observed a patchy distribution of jasmonates among streptophytes. We additionaly analyzed the corresponding culture and empty media to account for phytohormone excretion and environmental contamination. Extracellular auxins and cytokinins were frequently detected, while agar constituted a major external source of phenolic compounds. We provide a highly comprehensive evolution-directed screen of phytohormone compound occurrence and thoroughly discuss our data in the context of current plant hormonomics and phylogenomics. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/534998v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@1532f4org.highwire.dtl.DTLVardef@1c4396borg.highwire.dtl.DTLVardef@195d0eborg.highwire.dtl.DTLVardef@c71b11_HPS_FORMAT_FIGEXP M_FIG C_FIG

plant biology↗

bioRxiv2020

A role of DAO1 in oxidation of IAA amino acid conjugates revealed through metabolite, high throughput transcript and protein level analysis.

Auxin metabolism is, together with auxin transport, a key determinant of auxin signalling output in plant cells, yet details on the underlying mechanisms and factors involved are still largely unknown. Processes involved in the auxin metabolism are subject to regulation based on numerous signals, including auxin concentration itself. Altered auxin availability and the subsequent changes of auxin metabolite profiles can therefore elucidate the function and regulatory role of individual elements in the auxin metabolic machinery. After analysing auxin metabolism in auxin dependent tobacco BY-2 cell line grown in presence or absence of synthetic auxin 2,4-D we found that both conditions were similarly characterized by very low levels of endogenous indole-3-acetic acid (IAA) and its metabolites. However, metabolic profiling after exogenous application of IAA uncovered that the concentration of N-(2-oxindole-3-acetyl)-L-aspartic acid (oxIAA-Asp), the most abundantly formed auxin metabolite in the control culture, dramatically decreased in auxin-starved conditions. To describe the molecular mechanism behind this regulation, we analysed transcriptome and proteome changes caused by auxin starvation. While no changes in the expression of auxin biosynthetic machinery were observed, many genes related to auxin conjugation and degradation showed differential expression. Selected putative auxin glycosylating enzymes as well as members of the Gretchen Hagen 3 gene family involved in auxin amino acid conjugation showed both up- and down-regulation. Contrarily to that, all tobacco homologs of Arabidopsis thaliana DIOXYGENASE FOR AUXIN OXIDATION 1 (DAO1), known to be responsible for the formation of oxIAA from IAA, showed significant downregulation at both transcript and protein levels. To validate the role of DAO1 in auxin metabolism, we performed auxin metabolite profiling in BY-2 mutants carrying either siRNA-silenced or CRISPR-Cas9-mutated NtDAO1, as well as in dao1-1 Arabidopsis thaliana plants. Both mutants showed not only expectedly lower levels of oxIAA, but also significantly lower abundance of oxidated amino acid conjugates of IAA (oxIAA-Asp). Our results thus represent the first direct evidence on DAO1 activity on IAA amino acid conjugates. Statement of significanceHere we present an analysis of auxin metabolism on metabolite, transcript and protein levels in tobacco BY-2 cell line, collectively identifying oxidation of IAA amino acid conjugates as a new role of DIOXYGENASE FOR AUXIN OXIDATION 1 within an auxin-level-responsive metabolic system.

plant biology↗

bioRxiv2020

Mutually opposing activity of PIN7 splicing isoforms is required for auxin-mediated tropic responses in Arabidopsis thaliana

Advanced transcriptome sequencing has uncovered that the majority of eukaryotic genes undergo alternative splicing (AS). Nonetheless, little effort has been dedicated to investigating the functional relevance of particular splicing events, even those in the key developmental and hormonal regulators. Here we reveal, in the plant model Arabidopsis thaliana, that the PIN7 gene, which encodes a polarly localized transporter for the phytohormone auxin, produces two evolutionary-conserved transcripts. These isoforms PIN7a and PIN7b, differing in a 4 amino acid motif, are present at nearly equal levels in most cells. Although both variants do not differ in the subcellular localization and transport auxin with similar capacity, they closely associate and mutually influence their stability within the plasma membrane. Phenotypic complementation tests reveal that the functional contribution of PIN7b per se is minor but it markedly attenuates the prominent PIN7a activity, which is required for correct seedling apical hook formation and auxin-mediated tropic responses. These results establish alternative splicing of the PIN family as a conserved, functionally-relevant mechanism, unveiling an additional regulatory level of auxin-mediated plant development.

plant biology↗