Multi-class plant hormone HILIC-MS/MS analysis coupled with high-throughput phenotyping to investigate plant-environment interactions.

Plant hormones are chemical signals governing almost every aspect of a plant's life cycle and responses to environmental cues. They are enmeshed within complex signaling networks that can only be deciphered by using broad-scale analytical methods to capture information about several plant hormone classes simultaneously. Methods used for this purpose are all based on reversed-phase (RP) liquid chromatography and mass spectrometric detection. Hydrophilic interaction chromatography (HILIC) is an alternative chromatographic method that performs well in analyses of biological samples. We therefore developed and validated a HILIC method for broad-scale plant hormone analysis including a rapid sample preparation procedure; moreover, derivatization or fractionation is not required. The method enables plant hormone screening focused on polar and moderately polar analytes including cytokinins, auxins, jasmonates, abscisic acid and its metabolites, salicylates, indoleamines (melatonin), and 1-aminocyclopropane-1-carboxylic acid (ACC), for a total of 45 analytes. Importantly, the major pitfalls of ACC analysis have been addressed. Furthermore, HILIC provides orthogonal selectivity to conventional RP methods and displays greater sensitivity, resulting in lower limits of quantification. However, it is less robust, so procedures to increase its reproducibility were established. The method's potential is demonstrated in a case study by employing an approach combining hormonal analysis with phenomics to examine responses of three Arabidopsis ecotypes toward three abiotic stress treatments: salinity, low nutrient availability, and their combination. The case study showcases the value of the simultaneous determination of several plant hormone classes coupled with phenomics data when unraveling processes involving complex cross-talk under diverse plant-environment interactions.

Biochemical and structural basis of polyamine, lysine and ornithine acetylation catalyzed by spermine/spermidine N-acetyl transferase in moss and maize.

Polyamines such as spermidine and spermine are essential regulators of cell growth, differentiation, maintenance of ion balance and abiotic stress tolerance. Their levels are controlled by the spermidine/spermine N1 -acetyltransferase (SSAT) via acetylation to promote either their degradation or export outside the cell as shown in mammals. Plant genomes contain at least one gene coding for SSAT (also named NATA for N-AcetylTransferase Activity). Combining kinetics, HPLC-MS and crystallography, we show that three plant SSATs, one from the lower plant moss Physcomitrium patens and two from the higher plant Zea mays, acetylate various aliphatic polyamines and two amino acids lysine (Lys) and ornithine (Orn). Thus, plant SSATs exhibit a broad substrate specificity, unlike more specific human SSATs (hSSATs) as hSSAT1 targets polyamines, whereas hSSAT2 acetylates Lys and thiaLys. The crystal structures of two PpSSAT ternary complexes, one with Lys and CoA, the other with acetyl-CoA and polyethylene glycol (mimicking spermine), reveal a different binding mode for polyamine versus amino acid substrates accompanied by structural rearrangements of both the coenzyme and the enzyme. Two arginine residues, unique among plant SSATs, hold the carboxyl group of amino acid substrates. The most abundant acetylated compound accumulated in moss was N6 -acetyl-Lys, whereas N5 -acetyl-Orn, known to be toxic for aphids, was found in maize. Both plant species contain very low levels of acetylated polyamines. The present study provides a detailed biochemical and structural basis of plant SSAT enzymes that can acetylate a wide range of substrates and likely play various roles in planta.

Is the co-option of jasmonate signalling for botanical carnivory a universal trait for all carnivorous plants?

The carnivorous plants in the order Caryophyllales co-opted jasmonate signalling from plant defence to botanical carnivory. However, carnivorous plants have at least 11 independent origins, and here we ask whether jasmonate signalling has been co-opted repeatedly in different evolutionary lineages. We experimentally wounded and fed the carnivorous plants Sarracenia purpurea (order Ericales), Cephalotus follicularis (order Oxalidales), Drosophyllum lusitanicum (order Caryophyllales), and measured electrical signals, phytohormone tissue level, and digestive enzymes activity. Coronatine was added exogenously to confirm the role of jasmonates in the induction of digestive process. Immunodetection of aspartic protease and proteomic analysis of digestive fluid was also performed. We found that prey capture induced accumulation of endogenous jasmonates only in D. lusitanicum, in accordance with increased enzyme activity after insect prey or coronatine application. In C. follicularis, the enzyme activity was constitutive while in S. purpurea was regulated by multiple factors. Several classes of digestive enzymes were identified in the digestive fluid of D. lusitanicum. Although carnivorous plants from different evolutionary lineages use the same digestive enzymes, the mechanism of their regulation differs. All investigated genera use jasmonates for their ancient role, defence, but jasmonate signalling has been co-opted for botanical carnivory only in some of them.

A dual regulatory role for the arbuscular mycorrhizal master regulator RAM1 in tomato.

The REQUIRED FOR ARBUSCULAR MYCORRHIZATION1 (RAM1) transcription factor from the GRAS family is well known for its role as a master regulator of the arbuscular mycorrhizal (AM) symbiosis in dicotyledonous and monocotyledonous species, being essential in transcriptional reprogramming for the development and functionality of the arbuscules. In tomato, SlGRAS27 is the putative orthologue of RAM1 (here named SlRAM1), but has not yet been characterized. A reduced colonization of the root and impaired arbuscule formation were observed in SlRAM1-silenced plants, confirming the functional conservation of the RAM1 orthologue in tomato. However, unexpectedly, SlRAM1-overexpressing (UBIL:SlRAM1) plants also showed decreased mycorrhizal colonization. Analysis of non-mycorrhizal UBIL:SlRAM1 roots revealed an overall regulation of AM-related genes and a reduction of strigolactone biosynthesis. Moreover, external application of the strigolactone analogue GR244DO almost completely reversed the negative effects of SlRAM1 overexpression on the frequency of mycorrhization. However, it only partially recovered the pattern of arbuscule distribution observed in control plants. Our results strongly suggest that SlRAM1 has a dual regulatory role during mycorrhization and, in addition to its recognized action as a positive regulator of arbuscule development, it is also involved in different mechanisms for the negative regulation of mycorrhization, including the repression of strigolactone biosynthesis.

Comprehensive LC-MS/MS analysis of nitrogen-related plant metabolites.

We have developed and validated a novel LC-MS/MS method for simultaneously analyzing amino acids, biogenic amines, and their acetylated and methylated derivatives in plants. This method involves a one-step extraction of 2-5 mg of lyophilized plant material followed by fractionation of different biogenic amine forms, and exploits an efficient combination of hydrophilic interaction liquid chromatography (HILIC), reversed phase (RP) chromatography with pre-column derivatization, and tandem mass spectrometry (MS). This approach enables high-throughput processing of plant samples, significantly reducing the time needed for analysis and its cost. We also present a new synthetic route for deuterium-labeled polyamines. The LC-MS/MS method was rigorously validated by quantifying levels of nitrogen-related metabolites in seedlings of seven plant species, including Arabidopsis, maize, and barley, all of which are commonly used model organisms in plant science research. Our results revealed substantial variations in the abundance of these metabolites between species, developmental stages, and growth conditions, particularly for the acetylated and methylated derivatives and the various polyamine fractions. However, the biological relevance of these plant metabolites is currently unclear. Overall, this work contributes significantly to plant science by providing a powerful analytical tool and setting the stage for future investigations into the functions of these nitrogen-related metabolites in plants.

Stomatal effects and ABA metabolism mediate differential regulation of leaf and flower cooling in tomato cultivars exposed to heat and drought stress.

Co-occurring heat and drought stresses challenge crop performance. Stomata open to promote evaporative cooling during heat stress, but close to retain water during drought stress, which resulted in complex stomatal regulation under combined heat and drought. We aimed to investigate stomatal regulation in leaves and flowers of perennial, indeterminate cultivars of tomatoes subjected to individual and combined heat and drought stress followed by a recovery period, measuring morphological, physiological, and biochemical factors involved in stomatal regulation. Under stress, stomata of leaves were predominantly affected by drought, with lower stomatal density and stomatal closing, resulting in significantly decreased photosynthesis and higher leaf temperature. Conversely, stomata in sepals seemed affected mainly by heat during stress. The differential patterns in stomatal regulation in leaves and flowers persisted into the recovery phase as contrasting patterns in stomatal density. We show that flower transpiration is regulated by temperature, but leaf transpiration is regulated by soil water availability during stress. Organ-specific patterns of stomatal development and abscisic acid metabolism mediated this phenomenon. Our results throw light on the dual role of stomata in heat and drought tolerance of vegetative and generative organs, and demonstrate the importance of considering flower surfaces in the phenotyping of stomatal reactions to stress.

In vitro safety signals for potential clinical development of the anti-inflammatory pregnane X receptor agonist FKK6.

Based on the mimicry of microbial metabolites, functionalized indoles were demonstrated as the ligands and agonists of the pregnane X receptor (PXR). The lead indole, FKK6, displayed PXR-dependent protective effects in DSS-induced colitis in mice and in vitro cytokine-treated intestinal organoid cultures. Here, we report on the initial in vitro pharmacological profiling of FKK6. FKK6-PXR interactions were characterized by hydrogen-deuterium exchange mass spectrometry. Screening FKK6 against potential cellular off-targets (G protein-coupled receptors, steroid and nuclear receptors, ion channels, and xenobiotic membrane transporters) revealed high PXR selectivity. FKK6 has poor aqueous solubility but was highly soluble in simulated gastric and intestinal fluids. A large fraction of FKK6 was bound to plasma proteins and chemically stable in plasma. The partition coefficient of FKK6 was 2.70, and FKK6 moderately partitioned into red blood cells. In Caco2 cells, FKK6 displayed high permeability (A-B: 22.8 × 10-6 cm.s-1) and no active efflux. These data are indicative of essentially complete in vivo absorption of FKK6. The data from human liver microsomes indicated that FKK6 is rapidly metabolized by cytochromes P450 (t1/2 5 min), notably by CYP3A4. Two oxidized FKK6 derivatives, including DC73 (N6-oxide) and DC97 (C19-phenol), were detected, and these metabolites had 5-7 × lower potency as PXR agonists than FKK6. This implies that despite high intestinal absorption, FKK6 is rapidly eliminated by the liver, and its PXR effects are predicted to be predominantly in the intestines. In conclusion, the PXR ligand and agonist FKK6 has a suitable pharmacological profile supporting its potential preclinical development.

Exploring the Pharmacological Potential of Onosma riedliana: Phenolic Compounds and Their Biological Activities.

Onosma riedliana Binzet & Orcan, a traditionally used plant species, has been explored for its therapeutic potential in this study. The work presented here is the first report on the phenolic profile and biological activity of this species. Three extracts of varying polarity were prepared, with the methanolic extract containing the highest phenolic content (97.62 ± 0.20 mgGAE/g). Key phenolic compounds identified included pinoresinol, hesperidin, 4-hydroxybenzoic acid, and p-coumaric acid. The methanolic extract exhibited exceptional antioxidant properties, rivaling Trolox as a positive control, primarily attributed to hesperidin and luteolin. Moreover, the ethyl acetate extract demonstrated remarkable inhibition of cholinesterase and tyrosinase enzymes, while the methanolic extract displayed potent activity against carbohydrate hydrolytic enzymes, α-amylase and α-glucosidase. Again, phenolic compounds were shown to be responsible for the inhibition of cholinesterases and tyrosinase, but not for α-amylase and α-glucosidase. These findings underscore Onosma riedliana's potential for incorporation into diverse pharmaceutical formulations, given its multifaceted bioactivity.

Chilling induces sugar and ABA accumulation that antagonistically signals for symplastic connection of dormant potato buds.

Endodormancy (ED) is a crucial stage in the life cycle of many perennial plants. ED release requires accumulating a certain amount of cold exposure, measured as chilling units. However, the mechanism governing the effect of chilling on ED duration is poorly understood. We used the potato tuber model to investigate the response to chilling as associated with ED release. We measured the accumulation of specific sugars during and after chilling, defined as sugar units. We discovered that ED duration correlated better with sugar units accumulation than chilling units. A logistic function was developed based on sugar units measurements to predict ED duration. Knockout or overexpression of the vacuolar invertase gene (StVInv) unexpectedly modified sugar units levels and extended or shortened ED, respectively. Silencing the energy sensor SNF1-related protein kinase 1, induced higher sugar units accumulation and shorter ED. Sugar units accumulation induced by chilling or transgenic lines reduced plasmodesmal (PD) closure in the dormant bud meristem. Chilling or knockout of abscisic acid (ABA) 8'-hydroxylase induced ABA accumulation, in parallel to sweetening, and antagonistically promoted PD closure. Our results suggest that chilling induce sugar units and ABA accumulation, resulting in antagonistic signals for symplastic connection of the dormant bud.

polyamine uptake transporter 2 (put2) and decaying seeds enhance phyA-mediated germination by overcoming PIF1 repression of germination.

Red light promotes germination after activating phytochrome phyB, which destabilizes the germination repressor PIF1. Early upon seed imbibition, canopy light, unfavorable for photosynthesis, represses germination by stabilizing PIF1 after inactivating phyB. Paradoxically, later upon imbibition, canopy light stimulates germination after activating phytochrome phyA. phyA-mediated germination is poorly understood and, intriguingly, is inefficient, compared to phyB-mediated germination, raising the question of its physiological significance. A genetic screen identified polyamine uptake transporter 2 (put2) mutants that overaccumulate polyamines, a class of antioxidant polycations implicated in numerous cellular functions, which we found promote phyA-mediated germination. In WT seeds, our data suggest that canopy light represses polyamines accumulation through PIF1 while red light promotes polyamines accumulation. We show that canopy light also downregulates PIF1 levels, through phyA; however, PIF1 reaccumulates rapidly, which limits phyA-mediated germination. High polyamines levels in decaying seeds bypass PIF1 repression of germination and stimulate phyA-mediated germination, suggesting an adaptive mechanism promoting survival when viability is compromised.