Synthesis and biological activity of photostable and persistent abscisic acid analogs.

The plant hormone abscisic acid (ABA) plays a critical role in various environmental stress responses and has long been expected to be used in agriculture. However, the practical use of ABA has been limited, mainly because of its photoinstability and rapid biodegradation. We previously developed photostable ABA agonists, BP2A and Me 1',4'-trans-diol BP2A, in which the dienoic acid side chain of ABA was replaced with phenylacetic acid. This finding validated our structure-based approach in designing photostable agonists and provided a basis for developing a more potent or long-lasting ABA agonist. In this study, we synthesized novel BP2A analogs in which the cyclohexenone ring was modified to avoid catabolism by the ABA metabolic enzyme, ABA 8'-hydroxylase. All synthesized analogs showed higher photostability than BP2A under sunlight. In an Arabidopsis seed germination assay, (+)-compounds 5 and 6 with a tetralone ring displayed significantly stronger ABA agonist activity than (+)-BP2A. In contrast, in the in vitro phosphatase assays, both compounds showed comparable or weaker ABA receptor (PYL1) agonistic activity than (+)-BP2A, suggesting that the stronger ABA-like activity of (+)-5 and (+)-6 may arise from their metabolic stability in vivo. This study provides data relevant to designing photostable and persistent ABA agonists.

Molecular basis of the activation and dissociation of dimeric PYL2 receptor in abscisic acid signaling.

Phytohormone abscisic acid (ABA) is essential for plant responses to biotic and abiotic stresses. Dimeric receptors are a class of PYR1/PYL/RCAR (pyrabactin resistance 1/PYR1-like/regulatory component of ABA receptors) ABA receptors that are important for various ABA responses. While extensive experimental and computational studies have investigated these receptors, it remains not fully understood how ABA leads to their activation and dissociation for interaction with downstream protein phosphatase 2C (PP2C). Here, we study the activation and the homodimeric association processes of the PYL2 receptor as well as its heterodimeric association with protein phosphatase 2C 16 (HAB1) using molecular dynamics simulations. Free energy landscapes from ∼223 µs simulations show that dimerization substantially constrains PYL2 conformational plasticity and stabilizes the inactive state, resulting in lower ABA affinity. Also, we establish the thermodynamic model for competitive binding between homodimeric PYL2 association and heterodimeric PYL2-HAB1 association in the absence and presence of ABA. Our results suggest that the binding of ABA destabilizes the PYL2 complex and further stabilizes PYL2-HAB1 association, thereby promoting PYL2 dissociation. Overall, this study explains several key aspects on the activation of dimeric ABA receptors, which provide new avenues for selective regulation of these receptors.

Multi-Targeted Molecular Docking, Pharmacokinetics, and Drug-Likeness Evaluation of Okra-Derived Ligand Abscisic Acid Targeting Signaling Proteins Involved in the Development of Diabetes.

Diabetes mellitus is a global threat affecting millions of people of different age groups. In recent years, the development of naturally derived anti-diabetic agents has gained popularity. Okra is a common vegetable containing important bioactive components such as abscisic acid (ABA). ABA, a phytohormone, has been shown to elicit potent anti-diabetic effects in mouse models. Keeping its anti-diabetic potential in mind, in silico study was performed to explore its role in inhibiting proteins relevant to diabetes mellitus- 11ß-hydroxysteroid dehydrogenase (11ß-HSD1), aldose reductase, glucokinase, glutamine-fructose-6-phosphate amidotransferase (GFAT), peroxisome proliferator-activated receptor-gamma (PPAR-gamma), and Sirtuin family of NAD(+)-dependent protein deacetylases 6 (SIRT6). A comparative study of the ABA-protein docked complex with already known inhibitors of these proteins relevant to diabetes was compared to explore the inhibitory potential. Calculation of molecular binding energy (ΔG), inhibition constant (pKi), and prediction of pharmacokinetics and pharmacodynamics properties were performed. The molecular docking investigation of ABA with 11-HSD1, GFAT, PPAR-gamma, and SIRT6 revealed considerably low binding energy (ΔG from -8.1 to -7.3 Kcal/mol) and predicted inhibition constant (pKi from 6.01 to 5.21 µM). The ADMET study revealed that ABA is a promising drug candidate without any hazardous effect following all current drug-likeness guidelines such as Lipinski, Ghose, Veber, Egan, and Muegge.

Embryogenic callus induction from immature zygotic embryos and genetic transformation of Larix kaempferi 3x Larix gmelinii 9.

To date, there are few reports of the successful genetic transformation of larch and other conifers, mainly because it is difficult to transform and integrate exogenous genes. In this study, hybrid larch Larix kaempferi 3x Larix gmelinii 9 cones were collected on June 27, July 1, July 4, July 7 and July 16, 2017. Embryogenic callus induction was studied using a combination of different plant growth regulators and concentrations. The results showed that July 1 was the best stage; the highest induction rate was 10.83%, which cultured in BM medium (Button medium, which formula was listed in S1 Table) with 1.0 mg/L 2,4-D (2,4-dichlorophenoxyacetic acid) and 0.2 mg/L KT(kinetin). When cultured on a proliferation medium for 12 days, proliferation was the fastest, reaching 323.08%, which could also maintain the freshness and vitality. The suitable pre-culture medium for somatic embryogenesis was 1/4 BM medium containing 10 g/L inositol and 60 g/L sucrose. The combination of 45 mg/L ABA (abscisic acid) and 75 g/L PEG4000 (Polyethyene glycol 4000) could promote the number of somatic embryos, and reached the maximum, 210 140 per 1 g FW. The genetic transformation was carried out by the Agrobacterium-mediated transformation method with embryogenic callus cultured for 12 days. The results showed the optimal OD600 of the infection solution(suspension of A. tumefaciens) was 0.5, co-culture time was 2 days, and screening concentration of Hyg (hygromycin B) was 4 mg/L. In this study, the transformation rate of resistance callus was 32.1%. It provides a reference for low genetic transformation efficiency of larch at present. This study could be beneficial for the innovation and breeding of larch by genetic engineering and provides a certain basis for rapid propagation of excellent larch germplasm resources and genetic engineering breeding of larch and other conifers.

Effects of seed priming treatments on the germination and development of two rapeseed (Brassica napus L.) varieties under the co-influence of low temperature and drought.

The present study was performed to evaluate the effects of seed priming. This was done by soaking the seeds of two rapeseed cultivars, namely, ZY15 (tolerant to low temperature and drought) and HY49 (sensitive to low temperature and drought), for 12 h in varying solutions: distilled water, 138 mg/L salicylic acid (SA), 300 mg/L gibberellic acid (GA), 89.4 mg/L sodium nitroprusside (SNP), 3000 mg/L calcium chloride (CaCl2), and 30 mg/L abscisic acid (ABA). Primed and non-primed seeds were left to germinate at 15°C and -0.15 MPa (T15W15) and at 25°C and 0 MPa (T25W0), respectively. The results showed that SA, GA, SNP, CaCl2, and ABA significantly improved the germination potential (GP), germination rate (GR), germination index (GI), stem fresh weight (SFW), stem dry weight (SDW), root length (RL), stem length (SL), and seed vigor index (SVI) under T15W15. For ZY15 seeds under T25W0, GA, SNP, CaCl2, and ABA priming reduced the average germination time (96% after 5 days) compared to that of the control (88% after 5 days). For ZY15 seeds under T15W15, SA, SNP, CaCl2, and ABA priming, with respect to the control and water-treated groups, shortened the average germination time (92% after 5 days) compared to that of the control (80% after 5 days). For HY49 seeds under T25W0, GA, SNP, CaCl2, and ABA priming reduced the average germination time (92% after 5 days) compared to that of the control (85% after 5 days). Similarly, for HY49 seeds under T15W15, GA priming shortened the average germination time (89% after 5 days) compared to that of the control (83% after 5 days). These priming agents increased the net photosynthesis, stomatal conductivity, and transpiration rate of rape seedlings under conditions of low temperature and drought stress, while also decreasing intercellular carbon dioxide (CO2) concentrations. Additionally, SA, GA, SNP, CaCl2, and ABA increased superoxide dismutase concentrations (SOD) and ascorbic peroxidase (APX) activities of rape seedlings under stress conditions, while decreasing catalase (CAT) and peroxidase (POD) activities in ZY15 seedlings. In HY49, which is sensitive to low temperature and drought, all priming solutions, except for SNP, led to an increase in SOD activity levels and a decrease in CAT activity levels. Overall, SA, GA, SNP, and CaCl2 increased the concentrations of indoleacetic acid (IAA), GA, ABA, and cytokinin (CTK) in seedlings under stress conditions. Moreover, compared to SA, CaCl2, and ABA, GA (300 mg/L) and SNP (300 mol/L) showed improved priming effects for ZY15 and HY49 under stress conditions.

Uncovering the multi-level response of Glycine max L. to the application of allelopathic biostimulant from Levisticum officinale Koch.

The interest expressed by the agriculture in the category of innovative biostimulants is due to the intensive search for natural preparations. Our study is the first ever to report a complex approach to the use of allelopathic extracts from Levisticum officinale Koch. roots in soybean cultivation, includes analyses of morphological observations, and analyses of biochemical indicators. Hot method of aqueous extraction was applied. The extracts were administered via foliar application and soil treatment. Lovage extracts had high contents of polyphenolic compounds and rich micro- and macroelemental composition. The infusions did not contain gibberellic acid and indole-3-acetic acid but the abscisic acid and saccharose, glucose, and fructose were found. The extracts modified soybean plant physiology, as manifested by changes in biometric traits. Plants responded positively by increased yield. Seeds from the treated plants had higher contents of micro- and macroelements, as well as total concentrations of lipids (with a slight decrease in protein content). In addition, they featured changes in their amino acid profile and fatty acid composition. The application of allelopathic biostimulant caused increased concentrations of isoflavones and saponins. The natural biostimulants from Levisticum officinale may become a valuable tool in the sustainable agriculture.

Action of Multiple Rice ß-Glucosidases on Abscisic Acid Glucose Ester.

Conjugation of phytohormones with glucose is a means of modulating their activities, which can be rapidly reversed by the action of ß-glucosidases. Evaluation of previously characterized recombinant rice ß-glucosidases found that nearly all could hydrolyze abscisic acid glucose ester (ABA-GE). Os4BGlu12 and Os4BGlu13, which are known to act on other phytohormones, had the highest activity. We expressed Os4BGlu12, Os4BGlu13 and other members of a highly similar rice chromosome 4 gene cluster (Os4BGlu9, Os4BGlu10 and Os4BGlu11) in transgenic Arabidopsis. Extracts of transgenic lines expressing each of the five genes had higher ß-glucosidase activities on ABA-GE and gibberellin A4 glucose ester (GA4-GE). The ß-glucosidase expression lines exhibited longer root and shoot lengths than control plants in response to salt and drought stress. Fusions of each of these proteins with green fluorescent protein localized near the plasma membrane and in the apoplast in tobacco leaf epithelial cells. The action of these extracellular ß-glucosidases on multiple phytohormones suggests they may modulate the interactions between these phytohormones.

Knockout of Auxin Response Factor SlARF4 Improves Tomato Resistance to Water Deficit.

Auxin response factors (ARFs) play important roles in various plant physiological processes; however, knowledge of the exact role of ARFs in plant responses to water deficit is limited. In this study, SlARF4, a member of the ARF family, was functionally characterized under water deficit. Real-time fluorescence quantitative polymerase chain reaction (PCR) and ß-glucuronidase (GUS) staining showed that water deficit and abscisic acid (ABA) treatment reduced the expression of SlARF4. SlARF4 was expressed in the vascular bundles and guard cells of tomato stomata. Loss of function of SlARF4 (arf4) by using Clustered Regularly Interspaced Short Palindromic Repeats/Cas 9 (CRISPR/Cas 9) technology enhanced plant resistance to water stress and rehydration ability. The arf4 mutant plants exhibited curly leaves and a thick stem. Malondialdehyde content was significantly lower in arf4 mutants than in wildtype plants under water stress; furthermore, arf4 mutants showed higher content of antioxidant substances, superoxide dismutase, actual photochemical efficiency of photosystem II (PSII), and catalase activities. Stomatal and vascular bundle morphology was changed in arf4 mutants. We identified 628 differentially expressed genes specifically expressed under water deficit in arf4 mutants; six of these genes, including ABA signaling pathway-related genes, were differentially expressed between the wildtype and arf4 mutants under water deficit and unlimited water supply. Auxin responsive element (AuxRE) elements were found in these genes' promoters indicating that SlARF4 participates in ABA signaling pathways by regulating the expression of SlABI5/ABF and SCL3, thereby influencing stomatal morphology and vascular bundle development and ultimately improving plant resistance to water deficit.

Expression of AhATL1, an ABA Transport Factor Gene from Peanut, Is Affected by Altered Memory Gene Expression Patterns and Increased Tolerance to Drought Stress in Arabidopsis.

Arachis hypogaea abscisic acid transporter like-1 (AhATL1) modulates abscisic acid (ABA) sensitivity by specifically influencing the importing of ABA into cells, and is a key player in plant stress responses. However, there is limited information on ABA transporters in crops. In this study, we found that the level of AhATL1 expression and AhATL1 distribution increased more rapidly in the second drought (D2) compared with in the first drought (D1). Compared with the first recovery (R1), the AhATL1 expression level and ABA content remained at a higher level during the second recovery (R2). The heterologous overexpression of AhATL1 in Arabidopsis changed the expression pattern of certain memory genes and changed the post response gene type into the memory gene type. Regarding the proline and water content of Col (Arabidopsis thaliana L. Heynh., Col-0), atabcg22, and AhATL1-OX during drought training, the second drought (D2) was more severe than the first drought (D1), which was more conducive to maintaining the cell osmotic balance and resisting drought. In summary, drought stress memory resulted in a rapid increase in the AhATL1 expression and AhATL1 distribution level, and then raised the endogenous ABA content and changed the post response gene type into the memory gene type, which enhanced the drought resistance and recovery ability.

3'-(Phenyl alkynyl) analogs of abscisic acid: synthesis and biological activity of potent ABA antagonists.

We report here the synthesis and biological testing of 3'-(phenyl alkynyl) abscisic ABA analogs, a new class of potent ABA antagonists. These ABA analogs incorporate a rigid framework of eight carbon atoms attached at the 3'-carbon atom of ABA that prevents folding of the ABA analog-bound receptor required for ABA signalling. The two-step synthesis is based upon the optimized conversion of natural (S)-ABA to 3'-iodo ABA which can be coupled to phenyl acetylenes using Sonogashira conditions, or to styryl compounds through Suzuki chemistry. The parent 3'-(phenyl alkynyl) ABA analog 7 was obtained in 29% yield, 74% yield based on recovered starting material. In a lentil seed germination assay, compound 7 was found to have more potent activity than other known 3'-substituted ABA antagonists to date. In a structure activity study parasubstituted phenyl alkynyl analogs had comparable activity to the analog 7 while the 3'-styryl ABA 18 was only slightly less active. Analog 7 overcame ABA inhibition of germination and seedling growth in a wide range of mono and dicot plant species, including canola, lentil, soybean, rice, wheat, barley, cannabis and canary seed. 3'-(Phenyl alkynyl) ABA analogs have numerous potential practical agricultural applications including promoting ripening of crops, dormancy breaking of seeds and woody perennials, as well as promoting seed germination, and growth under stress conditions as demonstrated in this report.

Effect of RIP Overexpression on Abiotic Stress Tolerance and Development of Rice.

Ribosome-inactivating proteins (RIPs) are a class of cytotoxic enzymes that can inhibit protein translation by depurinating rRNA. Most plant RIPs are synthesized with a leader sequence that sequesters the proteins to a cell compartment away from the host ribosomes. However, several rice RIPs lack these signal peptides suggesting they reside in the cytosol in close proximity to the plant ribosomes. This paper aims to elucidate the physiological function of two nucleocytoplasmic RIPs from rice, in particular, the type 1 RIP referred to as OsRIP1 and a presumed type 3 RIP called nuRIP. Transgenic rice lines overexpressing these RIPs were constructed and studied for developmental effects resulting from this overexpression under greenhouse conditions. In addition, the performance of transgenic seedlings in response to drought, salt, abscisic acid and methyl jasmonate treatment was investigated. Results suggest that both RIPs can affect methyl jasmonate mediated stress responses.

AtTrxh3, a Thioredoxin, Is Identified as an Abscisic AcidBinding Protein in Arabidopsis thaliana.

Abscisic acid (ABA, 1) is a plant hormone that regulates various plant physiological processes such as seed developing and stress responses. The ABA signaling system has been elucidated; binding of ABA with PYL proteins triggers ABA signaling. We have previously reported a new method to isolate a protein targeted with a bioactive small molecule using a biotin linker with alkyne and amino groups, a protein cross-linker, and a bioactive small molecule with an azido group (azido probe). This method was used to identify the unknown ABA binding protein of Arabidopsis thaliana. As a result, AtTrxh3, a thioredoxin, was isolated as an ABA binding protein. Our developed method can be applied to the identification of binding proteins of bioactive compounds.

Engineering Lignin Nanomicroparticles for the Antiphotolysis and Controlled Release of the Plant Growth Regulator Abscisic Acid.

Lignin is the most abundant aromatic biopolymer in nature and is a major byproduct from the paper industry. The unlocking of lignin's potential for high-value applications has gained increasing attention in recent years. In this study, alkali lignin (AL), with a rigid conjugated structure and amphiphilic property, was used as a sustainable and eco-friendly encapsulation material for the protection and controlled release of photosensitive abscisic acid (ABA), an important and widely used plant growth regulator. Cetyltrimethylammonium bromide (CTAB) was used to induce the formation of AL-CTAB nanomicroparticles by self-assembly. The size and morphology of AL-CTAB particles were modified by changing the AL concentration and the dispersion agent. AL (0.3 M) dissolved in tetrahydrofuran could form a uniform size (300 nm) of particles with a regular spherical structure. Subsequently, ABA was loaded on the prepared nanomicroparticles to synthesize the capsule formulation of ABA@AL-CTAB. The controlled-release behavior and the antiphotolysis performance as well as the thermal stability of ABA@AL-CTAB were proved to be superior. Lasting inhibition of Arabidopsis and rice seed germination by ABA@AL-CTAB under light irradiations implied protection of ABA from photolysis. In addition, ABA@AL-CTAB could effectively regulate plant stomata, thereby increasing plant drought resistance. Overall, lignin is suitable for the preparation of agrochemical formulations with excellent controlled release and antiphotolysis performances.

Exogenous abscisic acid (ABA) promotes cadmium (Cd) accumulation in Sedum alfredii Hance by regulating the expression of Cd stress response genes.

Sedum alfredii Hance is a zinc (Zn) and cadmium (Cd) hyperaccumulator plant. However, the regulatory role of plant hormones in the Zn or Cd uptake and accumulation of S. alfredii remains unclear. In this work, the growth, Cd accumulation, abscisic acid (ABA) synthesis and catabolism, malonaldehyde (MDA) content, and transcriptional level of some Cd stress response genes under ABA and Cd co-treatment were investigated to reveal the impact of ABA on Cd resistance and Cd accumulation of S. alfredii. The results show that 0.2 mg/L ABA and 100 µmol/L Cd co-treatment enhanced Cd accumulation and growth in S. alfredii, whereas lower or higher ABA concentrations weaken or even reverse this effect, which was positively correlated with endogenous ABA content. The increase in endogenous ABA content might be the results of the increasing ABA synthetase activities and decreasing ABA lytic enzyme, which was induced by the application of 0.2 mg/L ABA under 100 µmol/L Cd treatment. Principal component analysis (PCA) indicated that ABA impacted the expression pattern of Cd stress response genes, which coincided with the Cd accumulation pattern in the shoots of S. alfredii. Cross-over analysis of partial least squares-discriminant analysis (PLS-DA) and correlation analysis indicated that HsfA4c, HMA4 expression in roots, and HMA2, HMA3, CAD, NAS expression in shoots were correlated with endogenous ABA, which suggests that endogenous ABA improves Cd resistance of seedlings, switches the root-to-shoot transporter from HMA2 to HMA4, and transports more Cd into apoplasts to promote Cd accumulation in the shoots of S. alfredii. Taken together, ABA plays an essential role not only in Cd resistance but also in Cd transport from root to shoot in S. alfredii under Cd stress.

Inoculation with abscisic acid (ABA)-catabolizing bacteria can improve phytoextraction of heavy metal in contaminated soil.

Promotion of plant capacity for accumulation of heavy metals (HMs) is one of the key strategies in enhancing phytoremediation in contaminated soils. Here we report that, Rhodococcus qingshengii, an abscisic acid (ABA)-catabolizing bacteria, clearly boosts levels of Cd, Zn, and Ni in wild-type Arabidopsis by 47, 24, and 30%, respectively, but no increase in Cu was noted, when compared with non-inoculated Arabidopsis plants in contaminated growth substrate. Furthermore, when compared with wild-type plants, R.qingshengii-induced increases in Cd, Zn, and Ni concentrations were more pronounced in abi1/hab1/abi2 (ABA-sensitive mutant) strains of Arabidopsis, whereas little effect was observed in snrk2.2/2.3 (ABA insensitive mutant). This demonstrates that metabolizing ABA might be indispensable for R. qingshengii to improve metal accumulation in plants. Bacterial inoculation significantly elevated the expression of Cd, Zn, and Ni-related transporters; whereas the transcript levels of Cu transporters remained unchanged. This result may be a reasonable explanation for why the uptake of Cd, Zn, and Ni in plants was stimulated by bacterial inoculation, while no effect was observed on Cu levels. From our results, we clearly demonstrate that R. qingshengii can increase the accumulation of Cd, Zn, and Ni in plants via an ABA-mediated HM transporters-associated mechanism. Metabolizing ABA in the plants by ABA-catabolizing bacterial inoculation might be an alternative strategy to improve phytoremediation efficiency in HMs contaminated soil.

A multi-channel localized surface plasmon resonance system for absorptiometric determination of abscisic acid by using gold nanoparticles functionalized with a polyadenine-tailed aptamer.

A multi-channel localized surface plasmon resonance system is described for absorptiometric determination of abscisic acid (ABA). The system is making use of gold nanoparticles and consists of a broadband light source, a multi-channel alignment device, and a fiber spectrometer. The method is based on the specific interaction between an ABA-binding aptamer and ABA. This induces the growth of gold nanoparticles (AuNPs) functionalized with a polyadenine-tailed aptamer that act as optical probes. Different concentrations of ABA give rise to varied morphologies of grown AuNPs. This causes a change of absorption spectra which is recorded by the system. ABA can be quantified by measurement of the peak wavelength shifts of grown AuNPs. Under optimized conditions, this method shows a linear relationship in the 1 nM to 10 µM ABA concentration range. The detection limit is 0.51 nM. The sensitivity of the ABA assay is strongly improved compared to the method based on salt-induced AuNP aggregation. This is attributed to the use of a poly-A-tailed aptamer and the catalytic ability of AuNPs. In the actual application, the ABA concentration of ABA in fresh leaves of rice is measured with the maximum relative error of 8.03% in comparison with the ELISA method. Graphical abstractSchematic representation of an absorptiometric approach for determination of abscisic acid based on the growth of polyA-tailed aptamer-AuNPs probes and a multi-channel localized surface plasmon resonance system.

Silicon and salicylic acid confer high-pH stress tolerance in tomato seedlings.

Alkalinity is a known threat to crop plant growth and production, yet the role of exogenous silicon (Si) and salicylic acid (SA) application has been largely unexplored. Here, we sought to understand the beneficial impacts of Si and SA on tomato seedlings during high-pH (9.0) stress. Results showed that Si- and SA-treated plants displayed higher biomass, chlorophyll contents, relative leaf water and better root system than none-treated plants under alkaline conditions. Both Si and SA counteracted the alkaline stress-induced oxidative damage by lowering the accumulation of reactive oxygen species and lipid peroxidation. The major antioxidant defence enzyme activities were largely stimulated by Si and SA, and these treatments caused significantly increased K+ and lowered Na+ concentrations in shoot and root under stress. Moreover, Si and SA treatments modulated endogenous SA levels and dramatically decreased abscisic acid levels in both shoot and root. Additionally, key genes involved in Si uptake, SA biosynthesis, the antioxidant defence system and rhizosphere acidification were up-regulated in Si and SA treatments under alkaline conditions. These results demonstrate that Si and SA play critical roles in improving alkaline stress tolerance in tomato seedlings, by modifying the endogenous Na+ and K+ contents, regulating oxidative damage and key genes and modulating endogenous hormone levels. These findings will help to broaden our understanding regarding the physiological and molecular mechanisms associated with the alkaline soil tolerance in plants.

Identifying new lead structures to enhance tolerance towards drought stress via high-throughput screening giving crops a quantum of solace.

Novel synthetic lead structures interacting with RCAR/(PYR/PYL) receptor proteins were identified based on the results of a high-throughput screening campaign of a large compound library followed by focused SAR studies of the three most promising hit clusters. Whilst indolinylmethyl sulfonamides 8y,z and phenylsulfonyl ethylenediamines 9y,z showed strong affinities for RCAR/ (PYR/PYL) receptor proteins in wheat, thiotriazolyl acetamides 7f,s exhibited promising efficacy against drought stress in vivo (wheat, corn and canola) combined with confirmed target interaction in wheat and arabidopsis thaliana. Remarkably, binding affinities of several representatives of 8 and 9 were on the same level or even better than the essential plant hormone abscisic acid (ABA).

Identification of recombinant AtPYL2, an abscisic acid receptor, in E. coli using a substrate-derived bioactive small molecule, a biotin linker with alkyne and amino groups, and a protein cross-linker.

Target protein identification of bioactive small molecules is one of the most important research in forward chemical genetics. The affinity chromatography technique to use a resin bound with a small molecule is often used for identification of a target protein of a bioactive small molecule. Here we report a new method to isolate a protein targeted with a bioactive small molecule using a biotin linker with alkyne and amino groups, protein cross-linker containing disulfide bond, and a bioactive small molecule with an azido group (azido probe). After an azido probe is associated with a target protein, the complex of a target protein and azido probe is covalently bound through the biotin linker by azide-alkyne Huisgen cycloaddition and protein cross-linker containing disulfide bond. This ternary complex is immobilized on an affinity matrix with streptavidin, and then the target protein is selectively eluted with a buffer containing a reducing agent for cleavage of disulfide bonds. This method uses a probe having an azido group, which a small functional group, and has the possibility to be a solution strategy to overcome the hindrance of a functional group introduced into the probe that reduces association a target protein. The effectiveness of the method in this study was shown using linker 1, 3'-azidoabscisic acid 3, and protein cross-linker containing a disulfide bond (DTSSP 5).

Abscisic Acid Standardized Fig (Ficus carica) Extracts Ameliorate Postprandial Glycemic and Insulinemic Responses in Healthy Adults.

Abscisic acid (ABA) can improve glucose homeostasis and reduce inflammation in mammals by activating lanthionine synthetase C-like 2 (LANCL2). This study examined the effects of two fig fruit extracts (FFEs), each administered at two different ABA doses, on glycemic index (GI) and insulinemic index (II) to a standard glucose drink. In a randomized, double-blind crossover study, 10 healthy adults consumed 4 test beverages containing FFE with postprandial glucose and insulin assessed at regular intervals over 2 h to determine GI and II responses. Test beverages containing 200 mg FFE-50× and 1200 mg FFE-10× significantly reduced GI values by -25% (P = 0.001) and -24% (P = 0.002), respectively. Two lower doses of FFE also reduced GI values compared with the reference drink (by approximately -14%), but the differences did not reach statistical significance. Addition of FFE to the glucose solution significantly reduced II values at all dosages and displayed a clear dose-response reduction: FFE-50× at 100 mg and 200 mg (-14% (P < 0.05) and -24% (P = 0.01), respectively) and FFE-10× at 600 mg and 1200 mg (-16% (P < 0.05) and -24% (P = 0.01), respectively). FFE supplementation is a promising nutritional intervention for the management of acute postprandial glucose and insulin homeostasis, and it is a possible adjunctive treatment for glycemic management of chronic metabolic disorders such as prediabetes and type 2 diabetes mellitus.