Biology subjects
Publications and source records attributed to Vondrakova, Z..
Jasmonic and salicylic acids are the major hormones involved in plant response to pests and pathogens. Here, we engineered autoluminescent plants that report activity of these hormones with up to 53-fold contrast. Using consumer-grade cameras, we imaged reporter Arabidopsis thaliana and Nicotiana benthamiana plants throughout normal development, and in response to attacks of pests and pathogens.
Plant development is under the morphogenic control of auxin. In addition to biosynthesis and metabolism, auxin concentration gradients are maintained by directional intercellular transport via PIN-FORMED (PIN) auxin efflux carriers. Although the structure-function properties of PINs have been described, it is still unclear whether individual members of PIN family could localize differently within the plasma membrane (PM) and whether their nanodomain distribution defines their function. To address this, we used cultured tobacco cells (Nicotiana tabacum L., cv. BY-2) and revealed by RNA-seq and RT-qPCR the cell stage-specific presence of transcripts of tobacco PIN homologs. We used high-resolution light microscopy and two independent immunoelectron microscopy techniques in cell lines expressing functional GFP-tagged inducible versions of NtPIN11T, NtPIN2T and NtPIN3bT. We show that NtPINs are distributed within specific PM nanodomains and that removal of the cell wall alters their appearance. By a series of in vivo microscopic observations, we provide evidence that NtPIN11T is the most homogenously distributed and the least mobile NtPIN. Pharmacological treatments suggested that the immobilization of NtPIN11T depends on the actin and microtubular cytoskeleton and the cell wall composition. Using comparative co-immunoprecipitation (co-IP) analysis of isolated membrane fractions for all three NtPINs we finally identified several novel interaction partners indicating a preferential association of NtPIN11T with cell wall GPI-anchored arabinogalactan proteins. In conclusion, our results suggest a model in which specific immobilization of PINs through interactions with the cell wall affects their function.
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.
The FLOWERING LOCUS T (FT) gene is the essential integrator of flowering regulatory pathways in angiosperms. The paralogs of the FT gene may perform antagonistic functions, as exemplified by BvFT1, that suppresses flowering in Beta vulgaris, unlike the paralogous activator BvFT2. The roles of FT genes in other amaranths were less investigated. Here, we transformed Arabidopsis thaliana with the FLOWERING LOCUS T like (FTL) genes of Chenopodium and found, that both FTL1 and FTL2-1 accelerated flowering, despite having been the homologs of the Beta vulgaris floral promoter and suppressor, respectively. The floral promotive effect of FTL2-1 was so strong that it caused lethality when overexpressed under the 35S promoter. FTL2-1 placed in inducible cassette accelerated flowering after the induction with methoxyphenozide. The occasional expression of FTL2-1 led to precocious flowering in some primary transformants even without chemical induction. After the FTL gene duplication in Amaranthaceae, the FTL1 copy maintained the role of floral activator. The second copy FTL2 underwent subsequent duplication and functional diversification, which enabled to control the onset of flowering in amaranths to adapt to variable environments. HIGHLIGHTThe FLOWERING LOCUS T like 2-1 (FTL 2-1) gene of Chenopodium acts as a strong activator of flowering in Arabidopsis, despite being a homolog of floral repressor BvFT1.
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.
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.