Uncovering the Interrelation between Metabolite Profiles and Bioactivity of In Vitro- and Wild-Grown Catmint (Nepeta nuda L.).

Nepeta nuda L. is a medicinal plant enriched with secondary metabolites serving to attract pollinators and deter herbivores. Phenolics and iridoids of N. nuda have been extensively investigated because of their beneficial impacts on human health. This study explores the chemical profiles of in vitro shoots and wild-grown N. nuda plants (flowers and leaves) through metabolomic analysis utilizing gas chromatography and mass spectrometry (GC-MS). Initially, we examined the differences in the volatiles' composition in in vitro-cultivated shoots comparing them with flowers and leaves from plants growing in natural environment. The characteristic iridoid 4a-α,7-ß,7a-α-nepetalactone was highly represented in shoots of in vitro plants and in flowers of plants from nature populations, whereas most of the monoterpenes were abundant in leaves of wild-grown plants. The known in vitro biological activities encompassing antioxidant, antiviral, antibacterial potentials alongside the newly assessed anti-inflammatory effects exhibited consistent associations with the total content of phenolics, reducing sugars, and the identified metabolic profiles in polar (organic acids, amino acids, alcohols, sugars, phenolics) and non-polar (fatty acids, alkanes, sterols) fractions. Phytohormonal levels were also quantified to infer the regulatory pathways governing phytochemical production. The overall dataset highlighted compounds with the potential to contribute to N. nuda bioactivity.

Influence of Exogenous 24-Epicasterone on the Hormonal Status of Soybean Plants.

Brassinosteroids (BRs) are key phytohormones involved in the regulation of major processes of cell metabolism that guide plant growth. In the past decades, new evidence has made it clear that BRs also play a key role in the orchestration of plant responses to many abiotic and biotic stresses. In the present work, we analyzed the impact of foliar treatment with 24-epicastasterone (ECS) on the endogenous content of major phytohormones (auxins, salicylic acid, jasmonic acid, and abscisic acid) and their intermediates in soybean leaves 7 days following the treatment. Changes in the endogenous content of phytohormones have been identified and quantified by LC/MS. The obtained results point to a clear role of ECS in the upregulation of auxin content (indole-3-acetic acid, IAA) and downregulation of salicylic, jasmonic, and abscisic acid levels. These data confirm that under optimal conditions, ECS in tested concentrations of 0.25 µM and 1 µM might promote growth in soybeans by inducing auxin contents. Benzoic acid (a precursor of salicylic acid (SA)), but not SA itself, has also been highly accumulated under ECS treatment, which indicates an activation of the adaptation strategies of cell metabolism to possible environmental challenges.

Non-specific effects of the CINNAMATE-4-HYDROXYLASE inhibitor piperonylic acid.

Chemical inhibitors are often implemented for the functional characterization of genes to overcome the limitations associated with genetic approaches. Although it is well established that the specificity of the compound is key to success of a pharmacological approach, off-target effects are often overlooked or simply neglected in a complex biological setting. Here we illustrate the cause and implications of such secondary effects by focusing on piperonylic acid (PA), an inhibitor of CINNAMATE-4-HYDROXYLASE (C4H) that is frequently used to investigate the involvement of lignin during plant growth and development. When supplied to plants, we found that PA is recognized as a substrate by GRETCHEN HAGEN 3.6 (GH3.6), an amido synthetase involved in the formation of the indole-3-acetic acid (IAA) conjugate IAA-Asp. By competing for the same enzyme, PA interferes with IAA conjugation, resulting in an increase in IAA concentrations in the plant. In line with the broad substrate specificity of the GH3 family of enzymes, treatment with PA increased not only IAA levels but also those of other GH3-conjugated phytohormones, namely jasmonic acid and salicylic acid. Finally, we found that interference with the endogenous function of GH3s potentially contributes to phenotypes previously observed upon PA treatment. We conclude that deregulation of phytohormone homeostasis by surrogate occupation of the conjugation machinery in the plant is likely a general phenomenon when using chemical inhibitors. Our results hereby provide a novel and important basis for future reference in studies using chemical inhibitors.

What determines root-sprouting ability: Injury or phytohormones?

PREMISE: Root-sprouting (RS) is an evolutionarily independent alternative to axillary stem branching for a plant to attain its architecture. Root-sprouting plants are better adapted to disturbance than non-RS plants, and their vigor is frequently boosted by biomass removal. Nevertheless, RS plants are rarer than plants that are not root-sprouters, possibly because they must overcome developmental barriers such as intrinsic phytohormonal balance or because RS ability is conditioned by injury to the plant body. The objective of this study was to identify whether phytohormones or injury enable RS. METHODS: In a greenhouse experiment, growth variables, root respiration, and phytohormones were analyzed in two closely related clonal herbs that differ in RS ability (spontaneously RS Inula britannica and rhizomatous non-RS I. salicina) with and without severe biomass removal. RESULTS: As previously reported, I. britannica is a root-sprouter, but injury did not boost its RS ability. Root respiration did not differ between the two species and decreased continuously with time irrespectively of injury, but their phytohormone profiles differed significantly. In RS species, the auxins-to-cytokinins ratio was low, and injury further decreased it. CONCLUSIONS: This first attempt to test drivers behind different plant growth forms suggests that intrinsic phytohormone regulation, especially the auxins-to-cytokinins ratio, might be behind RS ability. Injury, causing a phytohormonal imbalance, seems to be less important in spontaneously RS species than expected for RS species in general.

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

Advanced transcriptome sequencing has revealed 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. Combining approaches of genetics, biochemistry and advanced confocal microscopy, we describe the impact of alternative splicing on the PIN7 gene in the model plant Arabidopsis thaliana. PIN7 encodes a polarly localized transporter for the phytohormone auxin and produces two evolutionarily conserved transcripts, PIN7a and PIN7b. PIN7a and PIN7b, differing in a four amino acid stretch, exhibit almost identical expression patterns and subcellular localization. We reveal that they are closely associated and mutually influence each other's mobility within the plasma membrane. Phenotypic complementation tests indicate that the functional contribution of PIN7b per se is minor, but it markedly reduces the prominent PIN7a activity, which is required for correct seedling apical hook formation and auxin-mediated tropic responses. Our results establish alternative splicing of the PIN family as a conserved, functionally relevant mechanism, revealing an additional regulatory level of auxin-mediated plant development.

Hormonomic Changes Driving the Negative Impact of Broomrape on Plant Host Interactions with Arbuscular Mycorrhizal Fungi.

Belowground interactions of plants with other organisms in the rhizosphere rely on extensive small-molecule communication. Chemical signals released from host plant roots ensure the development of beneficial arbuscular mycorrhizal (AM) fungi which in turn modulate host plant growth and stress tolerance. However, parasitic plants have adopted the capacity to sense the same signaling molecules and to trigger their own seed germination in the immediate vicinity of host roots. The contribution of AM fungi and parasitic plants to the regulation of phytohormone levels in host plant roots and root exudates remains largely obscure. Here, we studied the hormonome in the model system comprising tobacco as a host plant, Phelipanche spp. as a holoparasitic plant, and the AM fungus Rhizophagus irregularis. Co-cultivation of tobacco with broomrape and AM fungi alone or in combination led to characteristic changes in the levels of endogenous and exuded abscisic acid, indole-3-acetic acid, cytokinins, salicylic acid, and orobanchol-type strigolactones. The hormonal content in exudates of broomrape-infested mycorrhizal roots resembled that in exudates of infested non-mycorrhizal roots and differed from that observed in exudates of non-infested mycorrhizal roots. Moreover, we observed a significant reduction in AM colonization of infested tobacco plants, pointing to a dominant role of the holoparasite within the tripartite system.

DIOXYGENASE FOR AUXIN OXIDATION 1 catalyzes the oxidation of IAA amino acid conjugates.

Together with auxin transport, auxin metabolism is a key determinant of auxin signaling output by plant cells. Enzymatic machinery involved in auxin metabolism is subject to regulation based on numerous inputs, including the concentration of auxin itself. Therefore, experiments characterizing altered auxin availability and subsequent changes in auxin metabolism could elucidate the function and regulatory role of individual elements in the auxin metabolic machinery. Here, we studied auxin metabolism in auxin-dependent tobacco BY-2 cells. We revealed that the concentration of N-(2-oxindole-3-acetyl)-l-aspartic acid (oxIAA-Asp), the most abundant auxin metabolite produced in the control culture, dramatically decreased in auxin-starved BY-2 cells. Analysis of the transcriptome and proteome in auxin-starved cells uncovered significant downregulation of all tobacco (Nicotiana tabacum) homologs of Arabidopsis (Arabidopsis thaliana) DIOXYGENASE FOR AUXIN OXIDATION 1 (DAO1), at both transcript and protein levels. Auxin metabolism profiling in BY-2 mutants carrying either siRNA-silenced or CRISPR-Cas9-mutated NtDAO1, as well as in dao1-1 Arabidopsis plants, showed not only the expected lower levels of oxIAA, but also significantly lower abundance of oxIAA-Asp. Finally, ability of DAO1 to oxidize IAA-Asp was confirmed by an enzyme assay in AtDAO1-producing bacterial culture. Our results thus represent direct evidence of DAO1 activity on IAA amino acid conjugates.

Heat Stress Targeting Individual Organs Reveals the Central Role of Roots and Crowns in Rice Stress Responses.

Inter-organ communication and the heat stress (HS; 45°C, 6 h) responses of organs exposed and not directly exposed to HS were evaluated in rice (Oryza sativa) by comparing the impact of HS applied either to whole plants, or only to shoots or roots. Whole-plant HS reduced photosynthetic activity (F v /F m and QY_Lss ), but this effect was alleviated by prior acclimation (37°C, 2 h). Dynamics of HSFA2d, HSP90.2, HSP90.3, and SIG5 expression revealed high protection of crowns and roots. Additionally, HSP26.2 was strongly expressed in leaves. Whole-plant HS increased levels of jasmonic acid (JA) and cytokinin cis-zeatin in leaves, while up-regulating auxin indole-3-acetic acid and down-regulating trans-zeatin in leaves and crowns. Ascorbate peroxidase activity and expression of alternative oxidases (AOX) increased in leaves and crowns. HS targeted to leaves elevated levels of JA in roots, cis-zeatin in crowns, and ascorbate peroxidase activity in crowns and roots. HS targeted to roots increased levels of abscisic acid and auxin in leaves and crowns, cis-zeatin in leaves, and JA in crowns, while reducing trans-zeatin levels. The weaker protection of leaves reflects the growth strategy of rice. HS treatment of individual organs induced changes in phytohormone levels and antioxidant enzyme activity in non-exposed organs, in order to enhance plant stress tolerance.

Cytokinin N-glucosides: Occurrence, Metabolism and Biological Activities in Plants.

Cytokinins (CKs) are a class of phytohormones affecting many aspects of plant growth and development. In the complex process of CK homeostasis in plants, N-glucosylation represents one of the essential metabolic pathways. Its products, CK N7- and N9-glucosides, have been largely overlooked in the past as irreversible and inactive CK products lacking any relevant physiological impact. In this work, we report a widespread distribution of CK N-glucosides across the plant kingdom proceeding from evolutionary older to younger plants with different proportions between N7- and N9-glucosides in the total CK pool. We show dramatic changes in their profiles as well as in expression levels of the UGT76C1 and UGT76C2 genes during Arabidopsis ontogenesis. We also demonstrate specific physiological effects of CK N-glucosides in CK bioassays including their antisenescent activities, inhibitory effects on root development, and activation of the CK signaling pathway visualized by the CK-responsive YFP reporter line, TCSv2::3XVENUS. Last but not least, we present the considerable impact of CK N7- and N9-glucosides on the expression of CK-related genes in maize and their stimulatory effects on CK oxidase/dehydrogenase activity in oats. Our findings revise the apparent irreversibility and inactivity of CK N7- and N9-glucosides and indicate their involvement in CK evolution while suggesting their unique function(s) in plants.

Publisher Correction: The lipid code-dependent phosphoswitch PDK1-D6PK activates PIN-mediated auxin efflux in Arabidopsis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The lipid code-dependent phosphoswitch PDK1-D6PK activates PIN-mediated auxin efflux in Arabidopsis.

Directional intercellular transport of the phytohormone auxin mediated by PIN-FORMED (PIN) efflux carriers has essential roles in both coordinating patterning processes and integrating multiple external cues by rapidly redirecting auxin fluxes. PIN activity is therefore regulated by multiple internal and external cues, for which the underlying molecular mechanisms are not fully elucidated. Here, we demonstrate that 3'-PHOSPHOINOSITIDE-DEPENDENT PROTEIN KINASE1 (PDK1), which is conserved in plants and mammals, functions as a molecular hub that perceives upstream lipid signalling and modulates downstream substrate activity through phosphorylation. Using genetic analysis, we show that the loss-of-function Arabidopsis pdk1.1 pdk1.2 mutant exhibits a plethora of abnormalities in organogenesis and growth due to defective polar auxin transport. Further cellular and biochemical analyses reveal that PDK1 phosphorylates D6 protein kinase, a well-known upstream activator of PIN proteins. We uncover a lipid-dependent phosphorylation cascade that connects membrane-composition-based cellular signalling with plant growth and patterning by regulating morphogenetic auxin fluxes.

Distinct metabolism of N-glucosides of isopentenyladenine and trans-zeatin determines cytokinin metabolic spectrum in Arabidopsis.

The diversity of cytokinin (CK) metabolites suggests their interconversions are the predominant regulatory mechanism of CK action. Nevertheless, little is known about their directionality and kinetics in planta. CK metabolite levels were measured in 2-wk-old Arabidopsis thaliana plants at several time points up to 100 min following exogenous application of selected CKs. The data were then evaluated qualitatively and by mathematical modeling. Apart from elevated levels of trans-zeatin (tZ) metabolites upon application of N6 -(Δ2 -isopentenyl)adenine (iP), we observed no conversions between the individual CK-types - iP, tZ, dihydrozeatin (DHZ) and cis-zeatin (cZ). In particular, there was no sign of isomerization between tZ and cZ families. Also, no increase of DHZ-type CKs was observed after application of tZ, suggesting low baseline activity of zeatin reductase. Among N-glucosides, those of iP were not converted back to iP while tZ N-glucosides were cleaved to tZ bases, thus affecting the whole metabolic spectrum. We present the first large-scale study of short-term CK metabolism kinetics and show that tZ N7- and N9-glucosides are metabolized in vivo. We thus refute the generally accepted hypothesis that N-glucosylation irreversibly inactivates CKs. The subsequently constructed mathematical model provides estimates of the metabolic conversion rates.