Cis-trans Research Articles

Design of Near-Infrared Emissive Molecular Switches Based on Stilbene-Appended Cyclometalated Bimetallic Ru(II)-Terpyridine Complexes.

In the present study, we have synthesized and thoroughly characterized two Ru(II) dimers with compositions [(ttpy)Ru(tpvpt')Ru(ttpy)](ClO4)3 and [(ttpy)Ru(t'pvpvpt')Ru(ttpy)](ClO4)2 incorporating phenylene-vinylene-substituted terpyridine bridging ligands capable of coordinating in both an NNN- and cyclometalated NNC-fashion. The complexes display strong absorption across the entire UV-vis spectral domain and exhibit luminescence in the NIR region (820-850 nm). The N atoms in the outer coordination sphere were employed for alteration of the photoredox behaviors of the complexes via acid-base equilibria. Decoordination of the Ru-C bonds in the presence of acid, followed by recoordination in the presence of base and heat, is also possible. Additionally, the phenylene-vinylene units in the bridging ligands facilitate trans → cis and trans-trans → trans-cis isomerization under visible light irradiation. The reverse isomerization (cis → trans and trans-cis → trans-trans) is also achieved upon UV light irradiation. Thus, "on-off" and "off-on" emission switching is facilitated through a judicious choice of external stimuli like acid, base, temperature, or light of particular wavelengths. Interestingly, the rate of photoswitching is significantly faster in the presence of acid compared to that in the absence of acid. DFT and TD-DFT calculations were also conducted to clearly visualize the electronic environment around the complex backbone and also for accurate assignment of the absorption and emission bands.

Optically controlled phase change wood for energy storage and heat release

Molecular solar thermal (MOST) fuels efficiently store solar energy and release it as heat by photoisomerization-induced phase change. However, MOSTs still struggle to meet practical demands due to its slow cis-trans isomerization rate, solvent-assisted charging and low energy storage density. Herein, we develop an optically controlled phase change wood (OCPCW) through impregnating methoxyazobenzene (mAZO) into delignified basswood with light energy storage and thermal energy release. Wood, as supporting frame, accelerates the rate of cis-trans isomerization due to the dispersion of mAZO by wood pores. In addition, the per unit storage energy density of mAZO in OCPCW can reach 307.5 J·g−1, which is higher than that of pure mAZO (229 J·g−1), due to the hydrogen bonding between cellulose and mAZO. Under sunlight irradiation, the cis-OCPCM undergoes isomerization to trans-OCPCM, releasing heat and leading to a temperature increase approximately 2 °C higher than that of delignified basswood under identical conditions. Furthermore, color changing in response to phase change, the OCPCM could be designed for encrypted and decrypted information with dual imaging modes through the color change and exothermic behavior. This work paves the way for the development of phase change wood for efficient solar energy storage and release and application in encrypted information display.

Design and synthesis of a novel curcumin-combretastatin A4 molecular skeleton: two pharmacophores.

The logical design and synthesis of a novel compound combretastatin A-4-integrated curcumin is presented. Claisen condensation of phenylacetone with ethyl acetates formed 1,5-diphenylpentane-2,4-dione. Condensation of the dione with benzaldehyde via a modified Pabon procedure formed combretastatin A-4-integrated curcumin. The single-crystal X-ray structure of one of the CA-4 integrated CURs was established as a representative example. Curcumin (CUR) and combretastatin A-4 (CA-4) are well-known bioactive natural products; however, their poor pharmacokinetic profiles and cis-trans isomerization under in vivo conditions, respectively, have limited their biological applications. Herein, coupling of an aryl group at the olefinic C2 and/or C6 position of CUR integrates a CA-4-like structure with cis-configuration locked to CUR. At the same time, aryl coupling created steric hindrance around the olefinic bond and could resist the reductive metabolism of CUR and contribute to a better pharmacokinetic profile. Remarkably, this modification did not disturb the functional groups in both the natural products (CUR and CA-4), which is promising for their therapeutic effects. Thus, the synthesized CA-4-integrated CUR molecular architecture offers a new molecular skeleton to be explored for bio-application.

Towards Bacterial Resistance via the Membrane Strategy: Enzymatic, Biophysical and Biomimetic Studies of the Lipid cis-trans Isomerase of Pseudomonas aeruginosa.

The lipid cis-trans isomerase (Cti) is a periplasmic heme-c enzyme found in several bacteria including Pseudomonas aeruginosa, a pathogen known for causing nosocomial infections. This metalloenzyme catalyzes the cis-trans isomerization of unsaturated fatty acids in order to rapidly modulate membrane fluidity in response to stresses that impede bacterial growth. As a consequence, breakthrough in the elucidation of the mechanism of this metalloenzyme might lead to new strategies to combat bacterial antibiotic resistance. We report the first comprehensive biochemical, electrochemical and spectroscopic characterization of a Cti enzyme. This has been possible by the successful purification of Cti from P. aeruginosa (Pa-Cti) in favorable yields with enzyme activity of 0.41 µmol/min/mg when tested with palmitoleic acid. Through a synergistic approach involving enzymology, site-directed mutagenesis, Raman spectroscopy, Mössbauer spectroscopy and electrochemistry, we identified the heme coordination and redox state, pinpointing Met163 as the sixth ligand of the FeII of heme-c in Pa-Cti. Significantly, the development of an innovative assay based on liposomes demonstrated for the first time that Cti catalyzes cis-trans isomerization directly using phospholipids as substrates without the need of protein partners, answering the important question about the substrate of Cti within the bacterial membrane.

PH and light-triggered shape transformation of block copolymer particles in emulsion droplets

Dual-responsive shape-transformable block copolymer (BCP) particles have arisen great attentions because of their tunable physical and chemical characteristics triggered by external stimuli. Herein, we developed a simple yet effective strategy to realize pH and light dual-triggered shape-switchable BCP particles through the co-assembly of BCPs with photo-responsive additive 4-hydroxyazobenzene (Azo-OH) within the evaporation emulsion droplets. The formation of hydrogen bonding interactions between the Azo-OH and 4-vinylpyridine (4VP) unit increases the volume fraction of P4VP domain, in turn leading to a morphological transition of the particles from onions to pupas by tuning the Azo-OH content. Subsequent light-induced trans–cis transition of the Azo unit induces the enhancement of the hydrophilicity of the P4VP domains, giving rise to internal shape transitions of the BCP particles from pupas to onions with P4VP at the outmost-layer. Furtherly, since the hydrogen bonds between Azo-OH and 4VP group are sensitive to pH, the interfacial properties of the emulsion droplets and the assembled structures are tuned through adjusting the environment pH of the aqueous solution, causing the formation of various particle shape, including pupas, raspberries, and onions. This work offers a promising method to engineer the microstructures of polymeric assemblies, which may attract more attention on the applications of the stimuli-responsive particles.

The conformational landscape of fold-switcher KaiB is tuned to the circadian rhythm timescale

How can a single protein domain encode a conformational landscape with multiple stably folded states, and how do those states interconvert? Here, we use real-time and relaxation-dispersion NMR to characterize the conformational landscape of the circadian rhythm protein KaiB from Rhodobacter sphaeroides. Unique among known natural metamorphic proteins, this KaiB variant spontaneously interconverts between two monomeric states: the "Ground" and "Fold-switched" (FS) states. KaiB in its FS state interacts with multiple binding partners, including the central KaiC protein, to regulate circadian rhythms. We find that KaiB itself takes hours to interconvert between the Ground and FS state, underscoring the ability of a single-sequence to encode the slow process needed for function. We reveal the rate-limiting step between the Ground and FS state is the cis-trans isomerization of three prolines in the fold-switching region by demonstrating interconversion acceleration by the prolyl isomerase Cyclophilin A. The interconversion proceeds through a "partially disordered" (PD) state, where the C-terminal half becomes disordered while the N-terminal half remains stably folded. We found two additional properties of KaiB's landscape. First, the Ground state experiences cold denaturation: At 4 °C, the PD state becomes the majorly populated state. Second, the Ground state exchanges with a fourth state, the "Enigma" state, on the millisecond-timescale. We combine AlphaFold2-based predictions and NMR chemical shift predictions to predict this Enigma state is a beta-strand register shift that relieves buried charged residues, and support this structure experimentally. These results provide mechanistic insight into how evolution can design a single-sequence that achieves specific timing needed for its function.

Sequence and structural determinants of RNAPII CTD phase-separation and phosphorylation by CDK7

The intrinsically disordered carboxy-terminal domain (CTD) of the largest subunit of RNA Polymerase II (RNAPII) consists of multiple tandem repeats of the consensus heptapeptide Y1-S2-P3-T4-S5-P6-S7. The CTD promotes liquid-liquid phase-separation (LLPS) of RNAPII in vivo. However, understanding the role of the conserved heptad residues in LLPS is hampered by the lack of direct biochemical characterization of the CTD. Here, we generated a systematic array of CTD variants to unravel the sequence-encoded molecular grammar underlying the LLPS of the human CTD. Using in vitro experiments and molecular dynamics simulations, we report that the aromaticity of tyrosine and cis-trans isomerization of prolines govern CTD phase-separation. The cis conformation of prolines and β-turns in the SPXX motif contribute to a more compact CTD ensemble, enhancing interactions among CTD residues. We further demonstrate that prolines and tyrosine in the CTD consensus sequence are required for phosphorylation by Cyclin-dependent kinase 7 (CDK7). Under phase-separation conditions, CDK7 associates with the surface of the CTD droplets, drastically accelerating phosphorylation and promoting the release of hyperphosphorylated CTD from the droplets. Our results highlight the importance of conformationally restricted local structures within spacer regions, separating uniformly spaced tyrosine stickers of the CTD heptads, which are required for CTD phase-separation.

Mechanism of methyl elaidate on the thermal oxidation behavior

The effects of cis–trans isomerization on the oxidation products of oleic acid (composites and structure characterization) were investigated. The reduction of thermal oxidation reaction of methyl elaidate (ME) was 6.72 % lower than that of methyl oleate (MO), and the oxidation products (core aldehydes) first increased and then decreased with the prolongation of thermal oxidation time. ME exhibited better oxidation stability than MO in free radicals, hydroperoxides, double bonds, aldehydes, and glycerides. The oxidation products were mainly 11-oxo-9-undecenoate (initial stage) and methyl 9-oxononanoate (later stage), following the free radical chain reaction mechanism. The H8 in double bond was more easily dehydrogenated in the chain initiation stage than H11, followed by the formation of core aldehyde through three intermediates and two transition states each pathway. ME required a higher energy barrier (1.3–13.6 kJ/mol) than MO reaction, making it more difficult for ME to undergo thermal oxidation reaction. The transition state 4 and 5 were rate determining steps of chain initiation reaction and proliferation reaction, respectively. This study was of great significance to further control the formation of these trans fats and their oxidation products.

Mesogenic cholesterol-naphthalene dimers: Synthesis, characterization, mesogenic properties, photochromic behaviour and theoretical insights

Synthesis and characterization of two new series of unsymmetrical dimers combining cholesterol with azo (Series I-n) or azomethine (Series II-n) naphthalene moieties were reported. The chemical structures were confirmed using FT-IR, 1H NMR, 13C NMR, mass spectrometry, and elemental analysis. Their thermotropic properties were investigated using POM, DSC and XRD. The analysis revealed that the first lower members (n = 1, 2) of each series lack mesogenic properties due to short, flexible spacers, while higher members (n = 3–5, 7, 9, 10) exhibit enantiotropic chiral nematic phases. Notably, higher members with odd spacers (n = 5, 7, 9) also display SmC* phases, highlighting the influence of spacer length and odd–even effects on molecular interactions. Dimers from series I-n demonstrated slightly higher clearing temperatures (TN-Iso) compared to series II-n. UV–Vis spectroscopy showed trans–cis isomerization, with a photoconversion efficiency of 56.7 % for the I-7 dimer. Computational studies using the B3LYP functional with a 6-31g (d, p) basis set provided insights into the electronic structure through electrostatic potential (ESP) and optical property analysis.

The unexpected and important role of alcohol stabilizer in thermal cis-trans isomerization of push-pull dyes in chloroform solution

The study demonstrates the influence of ethanol as a stabilizer in chloroform on the thermal cis-trans isomerization kinetics of photochromic compounds based on azobenzene and azopyridine derivatives. The significance of the addition of ethanol as a stabilizer in chloroform-based solutions on the isomerization reaction of the hydroxy- and the 6-hydroxylalkoxy-substituted azo compounds was investigated. The results have revealed that the presence of a polar stabilizer in a solvent can significantly reduce the reaction rate constant of the dark cis-trans isomerization due to the formation of hydrogen bonds at both ends of the azo-chromophore, thereby inhibiting the isomerization. Furthermore, a stabilizer can facilitate the reversion of azo-hydrazone tautomers of azophenols to their azobenzene form, resulting in a decrease in the reaction rate constant. The research presented in this paper can be essential for scientists who study the kinetics of the trans-cis and the cis-trans isomerization reactions of azo-chromophores. The results show that the use of solvents with polar stabilizers should be rather avoided, as their usage without information about stabilizers can lead to different experimental data. The authors should ensure that detailed information about stabilizers used in unstable solvents is included in the experimental section of their works (a detail currently often omitted in the literature).

Roles of ESIPT and TICT in the Photophysical Process of a Ratiometric Fluorescence Sensor for Al3.

Precise detection of Al3+ with the aid of a fluorescence sensor is of fundamental importance in the fields of water pollution control and food safety. A comprehensive understanding of the photophysical process of the sensor as well as its underlying detection mechanism is a precondition for the design of highly efficient sensors. This contribution performs a thorough investigation of the ratiometric fluorescence sensing mechanism of an Al3+ sensor with the aid of density functional theory and time-dependent density functional theory. Two excited-state intramolecular proton transfer (ESIPT) processes are observed on the S1 state potential energy surface, which leads to emission around 565 nm. A twisted intramolecular charge transfer state is observed after one of the ESIPT processes via cis-trans isomerization of the C═N bond. However, the large energy barrier hinders its occurrence, which is quite unusual. Al3+ is found to form three strong coordination bonds with the sensor, which eliminates ESIPT processes and induces a significant blue shift of the emission spectrum to 480 nm. The origination of the sensor's selectivity is also uncovered by investigating the interaction between the sensor and interfering metal ions.

X-ray Structure of Eleven New N,N′-Substituted Guanidines: Effect of Substituents on Tautomer Structure in the Solid State

Guanidine-containing molecules are an interesting class of compounds within both medicinal and material sciences. Having knowledge of their tautomerism is key in designing guanidines that interact with biological and chemical receptors. However, there are limited data on the solid-phase structure of N,N′-substituted guanidines. Thus, eleven guanidines bearing a 4,6-dimethylpyrimidyl at one end and substituents of varying sizes and electronic properties at the other side, were synthesised, crystallised, and analysed by X-ray crystallography. Calculations of isolated molecules of tautomer energies and bond lengths were performed for comparison. One class of guanidines crystallised as a cis–trans tautomer with the shorter bond directed towards the pyrimidyl unit. When more electron-deficient aniline substituents were inserted, the crystallised tautomer changed to a cis–cis form where the shorter bond was directed towards the aniline. The switch in the tautomer structure is concluded to be due to both the electronic properties of the substituents and the intermolecular hydrogen bonding in the crystal lattice.

Isomerization enhanced fluorescence brightness of benzobisthiadiazole-based NIR-II fluorophores for highly efficient fluorescence imaging: A theoretical perspective

As a cutting-edge technique, fluorescence imaging in the second near-infrared window (NIR-II) is vital for both biomedical research and clinical applications. However, its intravital imaging capacity has been restricted by the extremely limited brightness of NIR-II fluorophores. To address this challenge, we elucidated the inner mechanism of constructing high-performance NIR-II chromophores based on molecular isomer engineering from detailed computational investigations. Herein, three pairs of cis–trans isomers (cis-1, 2, 3 and trans-1, 2, 3) are designed by attaching amino, methoxyl and nitro moieties to different positions on the donor–acceptor-donor molecular skeleton with benzobisthiadiazole as the acceptor and triphenylamine as the donor. All the compounds feature efficient NIR-II emission ranging in 1000–1164 nm, and the photophysical characterizations are regulated by molecular isomer manipulation. Interestingly, fluorescence quantum yields of cis-isomers are higher than those of their trans-counterparts. These enhancements can be attributed to the significant reduction in non-radiative transition, as evidenced by the non-adiabatic excitation energy, non-adiabatic electron coupling and electron-vibration coupling. Meanwhile, fluorophores with nitro terminal group exhibit superior performance facilitated by the prominently intramolecular charge transfer. As a result, cis-3 achieves an optimal brightness maxima of 196.36 M−1 cm−1 at 632 nm. Notably, the energy gap and the hole-electron related H index are respectively identified as strongly relevant to the emission wavelength and brightness, making them capable of evaluating the feasibility of fluorophores as effective NIR-II candidates. These findings highlight the correlations between molecular geometry and luminescent properties, which will inspire more insights into the development of highly efficient NIR-II fluorophores through rational isomer engineering for biomedical applications.

Separation of the cis- and trans-isomers 1,4-cyclohexanedicarboxylic acid by a dynamic MOF

Achieving an efficient method to separate cis–trans isomers of trans-1,4-cyclohexanedicarboxylic acid remains a formidable challenge within the chemical industry. Here, we have synthesized a Co(II)-based dynamic metal–organic framework material involving the coordination of 1,4-cyclohexanedicarboxylic acid. Single-crystal X-ray diffraction and high-performance liquid chromatography (HPLC) analysis confirmed that only pure trans-1,4-cyclohexanedicarboxylic acid was present in all synthesized crystals, a characteristic that can be utilized for the separation of the cis- and trans-isomers of 1,4-cyclohexanedicarboxylic acid. Moreover, the MOF crystals displayed reversible structural changes and were capable of releasing the pure trans-isomer through ion exchange with a salt solution, underscoring their potential for applications in molecular separation.

Impact of dicarboxystilbene impurities on the properties of swift heavy ion latent tracks in PET films

We present a new hypothesis and supporting experimental evidence about the essential role played by small impurities of dicarboxystilbene in the process of UV treatment of PET films irradiated with swift heavy ions (SHI). This treatment both forms highly selective membranes with permeability values comparable to natural ones (the track-UV technique) and sharply accelerates the etching of latent tracks in membrane production (the track-etching technique). We hypothesize (1) that these high permeability value is due to the presence of a molecular motor of an Archimedes screw type in the central part of the latent track formed by photoinduced trans-cis isomerization of dicarboxystilbene molecules that are anchored in helical conformations in the latent track; and (2) that the acceleration of etching rates is due to the preferential accumulation of phenanthrene-type molecules in the central part of the latent track due to the existence of a cyclization channel for photoexcited molecules of cis-dicarboxystilbene. In the presence of alkali solution, phenanthrene molecules release electrons which behave as strong anions in aqueous solution, catalyzing alkaline hydrolysis of PET molecules in the central part of the track. We present experimental observations of photoinduced changes in transmission light intensity after track-UV light treatment of both pristine and SHI irradiated PET films that confirm the presence of trans-cis isomerization of dicarboxystilbene molecules and show their contribution to the formation of new helical conformations in SHI irradiated PET films during the light treatment.

The influence of bromine substitution and linking groups on the phase behaviour of light-responsive rod-like molecules

The effect of aromatic core bromination as well as different linking groups on the phase behaviour of light-responsive rod-like liquid crystalline materials is reported. For this purpose, two new series of laterally brominated rod-like molecules are designed and synthesized. The two series have three benzene rings connected using different linking groups either azo or ester units with different directions. Both series have pentyloxy chain at one end and an octyl chain at the other end in the first group or an octyloxy chain in the second group of materials. Additional lateral bromine substituent was inserted at ortho position with respect to the pentyloxy chain. The molecular self-assembly of the new materials was investigated using polarized optical microscope (POM), differential scanning calorimetry (DSC) and X-Ray diffraction (XRD). Depending on the type of the terminal chain (octyl or octyloxy) at one end and nature of the linking groups nematic phases as well as smectic C phases are observed, with variable temperature ranges. Interestingly, the nematic phases were found to exhibit cybotactic clusters with tilted SmC or orthogonal SmA structure (NCybC or NCybA) with wide temperature ranges. Finally, the isothermal photo switching because of cis–trans photoisomerization was investigated in solution as well as between different liquid crystal phases. This report could be of interest for optical information storage device applications.

Visualization of azobenzene mesogens trans-cis isomerization through structural color of photonic crystal

Azobenzene, a representative photoresponsive liquid crystal, has extensive applications in fields such as bionic robotics, actuators, artificial muscles, motors, and various functional devices. Photoisomerization (trans-cis isomerization) is the most important mechanism for its photoresponse. Currently, the isomerization of azobenzene mesogens can be measured through UV–vis absorption spectra, NMR techniques or transient grating methods. Visualization of the trans-cis isomerization of azobenzene mesogens still remains a challenge. In this study, a linear relationship is observed between the absorption intensity of azobenzene mesogens and the stopband shift of azobenzene inverse opals (AZOIOs) films during the isomerization process. This phenomenon can be used to determine the isomerization degree of azobenzene mesogens through the switching of the stopband of the AZOIOs film, enabling the visualization of the azobenzene mesogens’s configuration transition. The results of this study are highly significant for the design and fabrication of novel azobenzene devices.

Synthesis and characterization of rhenium and Technetium-99 m tricarbonyl and dicarbonyl complexes with quinaldic acid and Arsenic/Phosphorus derivatives

The synthesis and characterization of neutral tricarbonyl fac-[Re/99mTc(quin)(X)(CO)3] and dicarbonyl cis–trans-[Re/99mTc(quin)(X)2(CO)2] mixed ligand complexes with quinaldic acid (quin) as the bidentate ligand and trimethoxyphosphine [P(OCH3)3], tris(hydroxymethyl)-phosphine [P(CH2OH)3], triphenylphosphine (PPh3), and triphenylarsine (AsPh3) as the monodentate ligands (X) is described. The synthesis of the [2 + 1] tricarbonyl complexes proceeds by displacement of the water molecule of the fac-[Re/99mTc(quin)(H2O)(CO)3] by the monodentate ligand. Interestingly, the synthesis of the [2 + 1 + 1] dicarbonyl complexes was achieved only for PPh3 after replacing the CO group trans to PPh3 with a second PPh3 molecule. The latter complex was also obtained by refluxing quinaldic acid with the trans-mer-[Re(PPh3)2(Cl)(CO)3] precursor in toluene. Rhenium complexes were prepared in satisfactory yields and fully characterized. At the technetium-99 m level, complexes were produced in high radiochemical yields and characterized by comparative chromatographic analysis using the analogous rhenium complexes. Complexes have shown varying stability against transchelation by cysteine and histidine in agreement with the decreasing σ-donating capacity of the monodentate ligand (P(OCH3)3 > P(CH2OH)3 > PPh3 > AsPh3. The stable complexes with PPh3 showed high lipophilicity (LogP 2.90 and 3.10, respectively) due to the lipophilic nature of the monodentate ligands. The σ-donor and π-acceptor capacity of the monodentate ligand strongly influences the formation and stability of the complexes, and these characteristics should be considered before choosing the appropriate ligand for radiopharmaceuticals design.

Proline substitutions in the ASIC1 β11-12 linker slow desensitization

Desensitization is a prominent feature of nearly all ligand-gated ion channels. Acid-sensing ion channels (ASICs) undergo desensitization within hundreds of milliseconds to seconds upon continual extracellular acidification. The ASIC mechanism of desensitization is primarily due to the isomerization or “flipping” of a short linker joining the 11th and 12th β sheets in the extracellular domain. In the resting and active states this β11-12 linker adopts an “upward” conformation while in the desensitized conformation the linker assumes a “downward” state. It is unclear if a single linker adopting the downward state is sufficient to desensitize the entire channel, or if all three are needed or some more complex scheme. To accommodate this downward state, specific peptide bonds within the linker adopt either trans-like or cis-like conformations. Since proline-containing peptide bonds undergo cis-trans isomerization very slowly, we hypothesized that introducing proline residues in the linker may slow or even abolish ASIC desensitization, potentially providing a valuable research tool. Proline substitutions in the chicken ASIC1 β11-12 linker (L414P and Y416P) slowed desensitization decays approximately 100- to 1000-fold as measured in excised patches. Both L414P and Y416P shifted the steady-state desensitization curves to more acidic pH values while activation curves and ion selectivity were largely unaffected (except for a left-shifted activation pH50 of L414P). To investigate the functional stoichiometry of desensitization in the trimeric ASIC, we created families of L414P and Y416P concatemers with zero, one, two, or three proline substitutions in all possible configurations. Introducing one or two L414P or Y416P substitutions only slightly attenuated desensitization, suggesting that conformational changes in the single remaining faster wild-type subunits were sufficient to desensitize the channel. These data highlight the unusual cis-trans isomerization mechanism of ASIC desensitization and support a model where ASIC desensitization requires only a single subunit.

Selection of reference genes for expression profiling in biostimulation research of soybean

BackgroundPlant biostimulants constitute a promising environmentally friendly alternative for increasing crop yield and tolerance to unfavorable conditions. Among various types of such formulations, botanical extracts are gaining more recognition as products supporting plant performance. Moreover, novel tools such as cold-plasma or low-pressure microwave plasma discharge are being proposed as techniques that might improve their efficacy. Elucidation of the biostimulant’s mode of action requires complex research at a molecular level. Transcriptional changes occurring after biostimulant spraying might be investigated using RT-qPCR. However, this technique requires data normalization against stable endogenous controls.ResultsHere, we tested the expression stability of ten candidate genes in soybean plants exposed to various biostimulants treatment. Selection of the best-performing reference genes was conducted using four algorithms (geNorm, NormFinder, BestKeeper, and ΔCt method). According to the obtained results, Bic-C2 (RNA-binding protein Bicaudal-C) and CYP (cyclophilin type peptidyl-prolyl cis–trans isomerase) showed highest expression stability, while expression of EF1B (elongation factor 1-beta) fluctuated the most among a tested set of candidate genes.ConclusionsOverall, we recommend using Bic-C2 together with CYP for the RT-qPCR data normalization in soybean biostimulation experiments. To our best knowledge, this is the first comprehensive study of reference genes stability in plants subjected to biostimulant treatment. The results of this study will aid in further biostimulant research in crop plants, facilitating analyses performed on the transcriptional level.Graphical