N-Sulfonated IAA was discovered as a novel auxin metabolite in Urtica where it is biosynthesized de novo utilizing inorganic sulfate. It showed no auxin activity in DR5::GUS assay, implying possible inactivation/storage mechanism. A novel auxin derivative, N-sulfoindole-3-acetic acid (IAA-N-SO3H, SIAA), was discovered in stinging nettle (Urtica dioica) among 116 sulfonated metabolites putatively identified by a semi-targeted UHPLC-QqTOF-MS analysis of 23 plant/algae/fungi species. These sulfometabolites were detected based on the presence of a neutral loss of sulfur trioxide, as indicated by the m/z difference of 79.9568 Da in the MS2 spectra. The structure of newly discovered SIAA was confirmed by synthesizing its standard and comparing retention time, m/z and MS2 spectrum with those of SIAA found in Urtica. To study its natural occurrence, 73 species in total were further analyzed by UHPLC-QqTOF-MS or targeted UHPLC-MS/MS method with a limit of detection of 244 fmol/g dry weight. However, SIAA was only detected in Urtica at a concentration of 13.906 ± 9.603 nmol/g dry weight. Its concentration was > 30 times higher than that of indole-3-acetic acid (IAA), and the SIAA/IAA ratio was further increased under different light conditions, especially in continuous blue light. In addition to SIAA, structurally similar metabolites, N-sulfoindole-3-lactic acid, 4-(sulfooxy)phenyllactic acid and 4-(sulfooxy)phenylacetic acid, were detected in Urtica for the first time. SIAA was biosynthesized from inorganic sulfate in seedlings, as confirmed by the incorporation of exogenous 34S-ammonium sulfate (1 mM and 10 mM). SIAA exhibited no auxin activity, as demonstrated by both the Arabidopsis DR5::GUS assay and the Arabidopsis phenotype analysis. Sulfonation of IAA may therefore be a mechanism for IAA deactivation and/or storage in Urtica, similar to sulfonation of the jasmonates in Arabidopsis.

• Klíčová slova
• N-Sulfoindole-3-acetic acid, Indole-3-acetic acid, Mass spectrometry, Metabolomics, Phytohormone, Sulfonated,
• MeSH
• Arabidopsis metabolismus   MeSH
• kyseliny indoloctové * metabolismus   MeSH
• regulátory růstu rostlin metabolismus   MeSH
• tandemová hmotnostní spektrometrie MeSH
• Urtica dioica metabolismus   MeSH
• vysokoúčinná kapalinová chromatografie MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• indoleacetic acid MeSH Prohlížeč
• kyseliny indoloctové * MeSH
• regulátory růstu rostlin MeSH

Common centaury (Centaurium eryhtraea Rafn) is a medicinal plant species with vigorous morphogenic potential in vitro. The process of spontaneous shoot regeneration in a solid root culture is characteristic for this plant species. In this context, the aim of this work was to investigate the dynamic changes of endogenous phytohormones and carbohydrates content in root explants at different time points (0, 2, 4, 7, 14, 21, 28, and 60 days) during spontaneous centaury morphogenesis in vitro. Detailed analysis of cytokinins (CKs) showed that trans-zeatin (tZ) was the major bioactive CK at all time points. The corresponding riboside, tZ9R, was also determined in the majority of the identified transport forms, at all time-points. Further analysis of endogenous auxin revealed a significant increase in endogenous indole-3-acetic acid (IAA) after 21 days, when a huge jump in the ratio of IAA/bioactive CKs was also observed. The maximum total soluble sugar content was measured after 14 days, while a significant decrease was determined after 21 days, when the first regenerated adventitious shoots appeared. This undoubtedly indicates an increased energy requirement prior to the actual regeneration of the shoots. The obtained results indicate that the period from day 14 to day 21 involves the most dramatic disturbances in endogenous bioactive CKs, IAA and carbohydrate balance, which are very important and valuable factors for the onset of shoot regeneration.

• Klíčová slova
• auxin, centaury, cytokinin, morphogenesis, phytohormone, soluble sugars,
• Publikační typ
• časopisecké články MeSH

Auxin amino acid conjugates are considered to be storage forms of auxins. Previous research has shown that indole-3-acetyl-L-alanine (IAA-Ala), indole-3-propionyl-L-alanine (IPA-Ala) and indole-3-butyryl-L-alanine (IBA-Ala) affect the root growth of Brassica rapa seedlings. To elucidate the potential mechanism of action of the conjugates, we treated B. rapa seedlings with 0.01 mM IAA-, IPA- and IBA-Ala and investigated their effects on the auxin metabolome and transcriptome. IBA-Ala and IPA-Ala caused a significant inhibition of root growth and a decrease in free IAA compared to the control and IAA-Ala treatments. The identification of free auxins IBA and IPA after feeding experiments with IBA-Ala and IPA-Ala, respectively, confirms their hydrolysis in vivo and indicates active auxins responsible for a stronger inhibition of root growth. IBA-Ala caused the induction of most DEGs (807) compared to IPA-Ala (417) and IAA-Ala (371). All treatments caused similar trends in transcription profile changes when compared to control treatments. The majority of auxin-related DEGs were found after IBA-Ala treatment, followed by IPA-Ala and IAA-Ala, which is consistent with the apparent root morphology. In addition to most YUC genes, which showed a tendency to be downregulated, transcripts of auxin-related DEGs that were identified (UGT74E2, GH3.2, SAUR, IAA2, etc.) were more highly expressed after all treatments. Our results are consistent with the hypothesis that the hydrolysis of conjugates and the release of free auxins are responsible for the effects of conjugate treatments. In conclusion, free auxins released by the hydrolysis of all auxin conjugates applied affect gene regulation, auxin homeostasis and ultimately root growth inhibition.

• Klíčová slova
• Brassica rapa, amino acid auxin conjugates, auxin metabolome, indole-3-acetic acid, indole-3-butyric acid, indole-3-propionic acid, root growth inhibition, transcriptome,
• MeSH
• alanin MeSH
• Brassica rapa * genetika   MeSH
• indoly MeSH
• kyseliny indoloctové farmakologie   MeSH
• plži * MeSH
• semenáček genetika   MeSH
• transkriptom MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• alanin MeSH
• indoly MeSH
• kyseliny indoloctové MeSH

Photosynthesis is among the first processes negatively affected by environmental cues and its performance directly determines plant cell fitness and ultimately crop yield. Primarily sites of photosynthesis, chloroplasts are unique sites also for the biosynthesis of precursors of the growth regulator auxin and for sensing environmental stress, but their role in intracellular auxin homeostasis, vital for plant growth and survival in changing environments, remains poorly understood. Here, we identified two ATP-binding cassette (ABC) subfamily B transporters, ABCB28 and ABCB29, which export auxin across the chloroplast envelope to the cytosol in a concerted action in vivo. Moreover, we provide evidence for an auxin biosynthesis pathway in Arabidopsis thaliana chloroplasts. The overexpression of ABCB28 and ABCB29 influenced stomatal regulation and resulted in significantly improved water use efficiency and survival rates during salt and drought stresses. Our results suggest that chloroplast auxin production and transport contribute to stomata regulation for conserving water upon salt stress. ABCB28 and ABCB29 integrate photosynthesis and auxin signals and as such hold great potential to improve the adaptation potential of crops to environmental cues.

• Klíčová slova
• ABC transporter, auxin, drought, hormone transport, photosynthesis, salinity, stress,
• Publikační typ
• časopisecké články MeSH

BACKGROUND: Acidic phytohormones are small molecules controlling many physiological functions in plants. A comprehensive picture of their profiles including the active forms, precursors and metabolites provides an important insight into ongoing physiological processes and is essential for many biological studies performed on plants. RESULTS: A high-throughput sample preparation method for liquid chromatography-tandem mass spectrometry determination of 25 acidic phytohormones classed as auxins, jasmonates, abscisates and salicylic acid was optimised. The method uses a small amount of plant tissue (less than 10 mg fresh weight) and acidic extraction in 1 mol/L formic acid in 10% aqueous methanol followed by miniaturised purification on reverse phase sorbent accommodated in pipette tips organised in a 3D printed 96-place interface, capable of processing 192 samples in one run. The method was evaluated in terms of process efficiency, recovery and matrix effects as well as establishing validation parameters such as accuracy and precision. The applicability of the method in relation to the amounts of sample collected from distantly related plant species was evaluated and the results for phytohormone profiles are discussed in the context of literature reports. CONCLUSION: The method developed enables high-throughput profiling of acidic phytohormones with minute amounts of plant material, and it is suitable for large scale interspecies studies.

• Klíčová slova
• 3D printing, Evolutionarily distant plant species, High-throughput, In-tip microSPE, Liquid chromatography, Mass spectrometry, Miniaturisation, Plant hormones,
• Publikační typ
• časopisecké články MeSH

Soil salinization is increasing globally, driving a reduction in crop yields that threatens food security. Salinity stress reduces plant growth by exerting two stresses on plants: rapid shoot ion-independent effects which are largely osmotic and delayed ionic effects that are specific to salinity stress. In this study we set out to delineate the osmotic from the ionic effects of salinity stress. Arabidopsis thaliana plants were germinated and grown for two weeks in media supplemented with 50, 75, 100, or 125 mM NaCl (that imposes both an ionic and osmotic stress) or iso-osmolar concentrations (100, 150, 200, or 250 mM) of sorbitol, that imposes only an osmotic stress. A subsequent transcriptional analysis was performed to identify sets of genes that are differentially expressed in plants grown in (1) NaCl or (2) sorbitol compared to controls. A comparison of the gene sets identified genes that are differentially expressed under both challenge conditions (osmotic genes) and genes that are only differentially expressed in plants grown on NaCl (ionic genes, hereafter referred to as salt-specific genes). A pathway analysis of the osmotic and salt-specific gene lists revealed that distinct biological processes are modulated during growth under the two conditions. The list of salt-specific genes was enriched in the gene ontology (GO) term "response to auxin." Quantification of the predominant auxin, indole-3-acetic acid (IAA) and IAA biosynthetic intermediates revealed that IAA levels are elevated in a salt-specific manner through increased IAA biosynthesis. Furthermore, the expression of NITRILASE 2 (NIT2), which hydrolyses indole-3-acetonitile (IAN) into IAA, increased in a salt-specific manner. Overexpression of NIT2 resulted in increased IAA levels, improved Na:K ratios and enhanced survival and growth of Arabidopsis under saline conditions. Overall, our data suggest that auxin is involved in maintaining growth during the ionic stress imposed by saline conditions.

• Klíčová slova
• IAA, auxin, growth, ionic, osmotic, plant, salinity, salt stress,
• Publikační typ
• časopisecké články MeSH

The plant nucleus plays an irreplaceable role in cellular control and regulation by auxin (indole-3-acetic acid, IAA) mainly because canonical auxin signaling takes place here. Auxin can enter the nucleus from either the endoplasmic reticulum or cytosol. Therefore, new information about the auxin metabolome (auxinome) in the nucleus can illuminate our understanding of subcellular auxin homeostasis. Different methods of nuclear isolation from various plant tissues have been described previously, but information about auxin metabolite levels in nuclei is still fragmented and insufficient. Herein, we tested several published nucleus isolation protocols based on differential centrifugation or flow cytometry. The optimized sorting protocol leading to promising yield, intactness, and purity was then combined with an ultra-sensitive mass spectrometry analysis. Using this approach, we can present the first complex report on the auxinome of isolated nuclei from cell cultures of Arabidopsis and tobacco. Moreover, our results show dynamic changes in auxin homeostasis at the intranuclear level after treatment of protoplasts with free IAA, or indole as a precursor of auxin biosynthesis. Finally, we can conclude that the methodological procedure combining flow cytometry and mass spectrometry offers new horizons for the study of auxin homeostasis at the subcellular level.

• Klíčová slova
• auxin, auxin metabolism, flow cytometry, nucleus, subcellular fractionation,
• MeSH
• Arabidopsis účinky léků   metabolismus   ultrastruktura   MeSH
• buněčné jádro účinky léků   metabolismus   ultrastruktura   MeSH
• buněčné kultury MeSH
• centrifugace metody   MeSH
• frakcionace buněk přístrojové vybavení   metody   MeSH
• hmotnostní spektrometrie MeSH
• homeostáza fyziologie   MeSH
• indoly metabolismus   farmakologie   MeSH
• kyseliny indoloctové metabolismus   MeSH
• protoplasty chemie   MeSH
• průtoková cytometrie MeSH
• regulátory růstu rostlin metabolismus   MeSH
• rostlinné buňky účinky léků   metabolismus   ultrastruktura   MeSH
• tabák účinky léků   metabolismus   ultrastruktura   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• indole MeSH Prohlížeč
• indoly MeSH
• kyseliny indoloctové MeSH
• regulátory růstu rostlin MeSH

The endoplasmic reticulum (ER) is an extensive network of intracellular membranes. Its major functions include proteosynthesis, protein folding, post-transcriptional modification and sorting of proteins within the cell, and lipid anabolism. Moreover, several studies have suggested that it may be involved in regulating intracellular auxin homeostasis in plants by modulating its metabolism. Therefore, to study auxin metabolome in the ER, it is necessary to obtain a highly enriched (ideally, pure) ER fraction. Isolation of the ER is challenging because its biochemical properties are very similar to those of other cellular endomembranes. Most published protocols for ER isolation use density gradient ultracentrifugation, despite its suboptimal resolving power. Here we present an optimised protocol for ER isolation from Arabidopsis thaliana seedlings for the subsequent mass spectrometric determination of ER-specific auxin metabolite profiles. Auxin metabolite analysis revealed highly elevated levels of active auxin form (IAA) within the ER compared to whole plants. Moreover, samples prepared using our optimised isolation ER protocol are amenable to analysis using various "omics" technologies including analyses of both macromolecular and low molecular weight compounds from the same sample.

• Klíčová slova
• auxin, density gradient centrifugation, endoplasmic reticulum, mass spectrometry, subcellular fractionation,
• MeSH
• Arabidopsis cytologie   metabolismus   MeSH
• endoplazmatické retikulum metabolismus   MeSH
• kyseliny indoloctové metabolismus   MeSH
• metabolom MeSH
• metabolomika metody   MeSH
• proteiny huseníčku analýza   metabolismus   MeSH
• proteomika metody   MeSH
• rostlinné buňky MeSH
• semenáček cytologie   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• indoleacetic acid MeSH Prohlížeč
• kyseliny indoloctové MeSH
• proteiny huseníčku MeSH

Plants remodel their root architecture in response to a salinity stress stimulus. This process is regulated by an array of factors including phytohormones, particularly auxin. In the present study, in order to better understand the mechanisms involved in salinity stress adaptation in rice, we compared two contrasting rice cultivars-Luna Suvarna, a salt tolerant, and IR64, a salt sensitive cultivar. Phenotypic investigations suggested that Luna Suvarna in comparison with IR64 presented stress adaptive root traits which correlated with a higher accumulation of auxin in its roots. The expression level investigation of auxin signaling pathway genes revealed an increase in several auxin homeostasis genes transcript levels in Luna Suvarna compared with IR64 under salinity stress. Furthermore, protein profiling showed 18 proteins that were differentially regulated between the roots of two cultivars, and some of them were salinity stress responsive proteins found exclusively in the proteome of Luna Suvarna roots, revealing the critical role of these proteins in imparting salinity stress tolerance. This included proteins related to the salt overly sensitive pathway, root growth, the reactive oxygen species scavenging system, and abscisic acid activation. Taken together, our results highlight that Luna Suvarna involves a combination of morphological and molecular traits of the root system that could prime the plant to better tolerate salinity stress.

• Klíčová slova
• PIN, YUCCA, abiotic stress, auxin, mass spectrometry, proteomics, rice, root, salinity,
• Publikační typ
• časopisecké články MeSH

Embryogenesis in seed plants is the process during which a single cell develops into a mature multicellular embryo that encloses all the modules and primary patterns necessary to build the architecture of the new plant after germination. This process involves a series of cell divisions and coordinated cell fate determinations resulting in the formation of an embryonic pattern with a shoot-root axis and cotyledon(s). The phytohormone auxin profoundly controls pattern formation during embryogenesis. Auxin functions in the embryo through its maxima/minima distribution, which acts as an instructive signal for tissue specification and organ initiation. In this review, we describe how disruptions of auxin biosynthesis, transport, and response severely affect embryo development. Also, the mechanism of auxin action in the development of the shoot-root axis and the three-tissue system is discussed with recent findings. Biological tools that can be implemented to study the auxin function during embryo development are presented, as they may be of interest to the reader.

• MeSH
• biologický transport MeSH
• kořeny rostlin růst a vývoj   MeSH
• kyseliny indoloctové metabolismus   MeSH
• regulátory růstu rostlin metabolismus   MeSH
• semena rostlinná růst a vývoj   MeSH
• signální transdukce MeSH
• výhonky rostlin růst a vývoj   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• kyseliny indoloctové MeSH
• regulátory růstu rostlin MeSH