Heat-stress-responsive HvHSFA2e gene regulates the heat and drought tolerance in barley through modulation of phytohormone and secondary metabolic pathways.

KEY MESSAGE: The heat stress transcription factor HSFA2e regulates both temperature and drought response via hormonal and secondary metabolism alterations. High temperature and drought are the primary yield-limiting environmental constraints for staple food crops. Heat shock transcription factors (HSF) terminally regulate the plant abiotic stress responses to maintain growth and development under extreme environmental conditions. HSF genes of subclass A2 predominantly express under heat stress (HS) and activate the transcriptional cascade of defense-related genes. In this study, a highly heat-inducible HSF, HvHSFA2e was constitutively expressed in barley (Hordeum vulgare L.) to investigate its role in abiotic stress response and plant development. Transgenic barley plants displayed enhanced heat and drought tolerance in terms of increased chlorophyll content, improved membrane stability, reduced lipid peroxidation, and less accumulation of ROS in comparison to wild-type (WT) plants. Transcriptome analysis revealed that HvHSFA2e positively regulates the expression of abiotic stress-related genes encoding HSFs, HSPs, and enzymatic antioxidants, contributing to improved stress tolerance in transgenic plants. The major genes of ABA biosynthesis pathway, flavonoid, and terpene metabolism were also upregulated in transgenics. Our findings show that HvHSFA2e-mediated upregulation of heat-responsive genes, modulation in ABA and flavonoid biosynthesis pathways enhance drought and heat stress tolerance.

PRpnp, a novel dual activity PNP family protein improves plant vigour and confers multiple stress tolerance in Citrus aurantifolia.

Under field conditions, plants are often simultaneously exposed to several abiotic and biotic stresses resulting in significant reductions in growth and yield; thus, developing a multi-stress tolerant variety is imperative. Previously, we reported the neofunctionalization of a novel PNP family protein, Putranjiva roxburghii purine nucleoside phosphorylase (PRpnp) to trypsin inhibitor to cater to the needs of plant defence. However, to date, no study has revealed the potential role and mechanism of either member of this protein group in plant defence. Here, we overexpressed PRpnp in Citrus aurantifolia which showed nuclear-cytoplasmic localization, where it functions in maintaining the intracellular purine reservoir. Overexpression of PRpnp significantly enhanced tolerance to salt, oxidative stress, alkaline pH, drought and two pests, Papilio demoleus and Scirtothrips citri in transgenic plants. Global gene expression studies revealed that PRpnp overexpression up-regulated differentially expressed genes (DEGs) related to ABA- and JA-biosynthesis and signalling, plant defence, growth and development. LC-MS/MS analysis validated higher endogenous ABA and JA accumulation in transgenic plants. Taken together, our results suggest that PRpnp functions by enhancing the endogenous ABA and JA, which interact synergistically and it also inhibits trypsin proteases in the insect gut. Also, like other purine salvage genes, PRpnp also regulates CK metabolism and increases the levels of CK-free bases in transgenic Mexican lime. We also suggest that PRpnp can be used as a potential candidate to develop new varieties with improved plant vigour and enhanced multiple stress resistance.

Efficient transformation and regeneration of transgenic plants in commercial cultivars of Citrus aurantifolia and Citrus sinensis.

Citrus is one of the major horticultural crops with high economic and nutraceutical value. Despite the fact that conventional research has developed numerous improved varieties, citriculture is still susceptible to various stresses and requires innovative solutions such as genetic engineering. Among all the currently available modern approaches, Agrobacterium-mediated transformation is the most efficient method for introducing desired traits in citrus. However, being a non-host for Agrobacterium, various citrus species, including Citrus aurantifolia and Citrus sinensis, are recalcitrant to this method. The available reports on Agrobacterium-mediated transformation of commercial citrus cultivars show very low transformation efficiency with poor recovery rates of whole transgenic plantlets. Here, we provide an efficient and reliable procedure of Agrobacterium-mediated transformation for both C. aurantifolia and C. sinensis. This protocol depends on providing callus-inducing treatment to explants before and during Agrobacterium co-cultivation, using optimum conditions for shoot regeneration and modifying in-vitro micrografting protocol to combat the loss of transgenic lines. As transgenic citrus shoots are difficult to root, we also developed the ideal conditions for their rooting. Using this protocol, the whole transgenic plantlets of C. aurantifolia and C. sinensis can be developed in about ~ 4 months, with transformation efficiency of 30% and 22% for the respective species.

The enhanced phosphorus use efficiency in phosphate-deficient and mycorrhiza-inoculated barley seedlings involves activation of different sets of PHT1 transporters in roots.

MAIN CONCLUSION: Transcriptional activation of subfamily II PHT1 members in roots is associated with the enhanced phosphorus use efficiency and growth promotion of barley seedlings inoculated with Glomus species. The arbuscular mycorrhizal (AM) fungi symbiotic associations in cereal crops are known to regulate growth in cultivar-specific manner and induce phosphate (Pi) transporters (PHT1) in roots. In the present study, we observed that both AM colonization of roots by Glomus species and phosphate starvation enhanced phosphorus use efficiency (PUE) in barley seedlings. Our search for the full complement of PHT1 members in the recently sequenced barley genome identified six additional genes, totaling their number to 17. Both AM colonization and Pi starvation triggered activation of common as well as different PHT1s. Pi starvation led to the robust upregulation of HvPHT1;6.2/6.3 at 7d and weak activation of HvPHT1;1 in shoots at 3d time-point. In roots, only HvPHT1;1, HvPHT1;6.2/6.3, HvPHT1;7, HvPHT1;8, HvPHT1;11.2 and HvPHT12 were induced at least one of the time-points. AM colonization specifically upregulated HvPHT1;11, HvPHT1;11.2, HvPHT1;12 and HvPHT1;13.1/13.2, members belonging to subfamily II, in roots. Sucrose availability seems to be obligatory for the robust activation of HvPHT1;1 as unavailability of this metabolite generally weakened its upregulation under Pi starvation. Intriguingly, lack of sucrose supply also led to induction of HvPHT1;5, HvPHT1;8, and HvPHT1;11.2 in either roots or shoot or both. The mRNA levels of HvPHT1;5 and HvPHT1;11.2 were not severely affected under combined deficiency of Pi and sucrose. Taken together, this study not only identify additional PHT1 members in barley, but also ascertain their AM, Pi and sucrose-specific transcript accumulation. The beneficial role of AM fungi in the promotion of PUE and barley seedlings' growth is also demonstrated.

Novel Curcumin-Resveratrol Solid Nanoparticles Synergistically Inhibit Proliferation of Melanoma Cells.

Polyphenols such as curcumin (Cur) and resveratrol (Res) have been recently shown to have potential to inhibit proliferation of highly aggressive melanoma cells. This study was designed to investigate the feasibility of a topical delivery system, using a solid lipid nanoparticles (SLNs) loaded delivery systems, that can enhance the skin penetration and anti-cancer efficacy of combination of these polyphenols. Negatively charged Cur-Res SLNs with a mean diameter of 180.2 ± 7.7 nm were prepared using high shear homogenization method. Cur-Res SLNs were found to be stable up to 2 weeks under 4°C. The in vitro release study showed that Res was released five time more than curcumin. The permeability of resveratrol was about 1.67 times that of curcumin from the SLN-gel formulation which was significantly (p < 0.05) lower than from SLN suspension. More than 70% of Cur-Res SLNs were bound to skin locally in a skin binding study suggesting potentially utility of Cur-Res SLNs in the treatment of localized melanoma. In fact, the electrical cell-substrate impedance sensing (ECIS) measurements suggested that Cur-Res combination has potential to stop cell migration of B16F10 melanoma cells. Furthermore, both, Cur-Res SLNs and Cur-Res solution at the ratio of 3:1 demonstrated a strong synergistic inhibition of SK-MEL-28 melanoma cell proliferation. Further evaluation of Cur-Res SLNs in vivo melanoma models are warranted to establish the clinical utility of Cur-Res formulations in melanoma therapy.

Ternary solid dispersions: classification and formulation considerations.

The number of active pharmaceutical compounds from the biopharmaceutical classification system (BCS) belonging to Class II and IV have significantly increased in recent years. These compounds have high therapeutic potential but are difficult to formulate as oral dosage forms due to their poor aqueous solubility. The solubility and bioavailability of these poorly water-soluble compounds can be increased by various formulation approaches, such as amorphous solid dispersions (ASD), salt formation, complexations, etc. Out of these techniques, the ASD approach, where compounds are converted into amorphous form and embedded in the hydrophilic matrix, have been successfully used in many marketed preparations. The recent advancement of this ASD approach is the design of ternary solid dispersions (TSD), where an additional component is added to further improve their performance in terms of solubility, stability, and processability. This review discusses the classification, mechanism of performance improvement, preparation techniques, and characterizations for TSD.

Investigating crystallization tendency, miscibility, and molecular interactions of drug-polymer systems for the development of amorphous solid dispersions.

Crystallization tendencies, thermal analysis [i.e. glass transition temperature (Tg)], crystallinity, and melting point depression, along with theoretical calculations such as solubility parameter, of five different drugs [i.e. curcumin (CUR), indomethacin (IND), flutamide (FLU), dipyridamole (DIP), and griseofulvin (GRI)] in the absence and presence of four different polymers in various drug-polymer ratios were determined and analyzed. Physical states of the drug in the solid dispersions (SDs) and their stability were characterized by X-ray diffraction and modulated differential scanning calorimetry. Infrared (IR) and Raman were used in selected systems (i.e. CUR, DIP, and GRI systems) to explore the role of drug-polymer interactions in the amorphization of SDs. The crystallization tendencies of pure drugs were categorized as low (CUR, IND), moderate (FLU), and high (DIP, GRI). In the presence of selected polymers, the crystallization tendency of the drugs changed, though a high polymer concentration was required for high crystallization-tendency drugs [i.e. DIP and GRI (>50% w/w)]. Polymers showing a greater effect on the crystallization tendency of drugs were found to have higher drug-polymer miscibility and stronger molecular interactions. Drug-polymer systems selected from the investigation of physical mixtures formed stable amorphous solid dispersions (ASD). Furthermore, the rank order of the crystallization tendency of drug-polymer systems correlated well with those on miscibility and molecular interactions. Those rank orders also correlated well with the stability of prepared/reported SDs. Hence, the developed approach has significant potential to be a rational screening method for the development of amorphous SDs.

Characterization and Systemic Delivery of Dibenzoylmethane via the Intranasal Route.

Intranasal (IN) administration is known to be noninvasive with the potential to carry a drug or vaccine directly to the blood, bypassing first-pass metabolism in the liver and the harsh environment of the gastrointestinal system. Orally administered dibenzoylmethane (DBM) has been shown experimentally to be neuroprotective in animal models of tauopathy and prion disease and effective in the treatment of certain forms of cancers. The purpose of this study was to prepare, characterize, and test formulations of DBM designed for IN administration. DBM was formulated in brain homogenate (BH) and hypromellose and as nanoparticles (NPs). These formulations were detected using UPLC and characterized in solid and suspension states; NPs were also characterized by in vitro cell culture-based studies. Particle size for DBM NP was 163.8 ± 3.2 nm, and in vitro release studies showed 95.80% of DBM was released from the NPs within 8 days. In vitro cell, culture studies suggested no drug uptake until 6 h. A histological analysis of nasal cavity (NC) sections and blood detection studies were carried out 30 min after inhalation. DBM amounting to 40.77 ± 4.93 and 44.45 ± 5.36 ng/mL was detected in the blood of animals administered DBM in polymeric and NP formulation, respectively. Histological studies on NCs confirmed the presence of BH within lymphatic vessels in the lamina propria of each animal; BH was identified traversing the mucosa in 2 animals. Thus, formulations for DBM administered via IN route were successfully designed and characterized and able to cross the nasal mucosa following inhalation.

Overexpression of wheat transcription factor (TaHsfA6b) provides thermotolerance in barley.

MAIN CONCLUSION: Overexpressing a heat shock factor gene (TaHsfA6bT) from wheat provides thermotolerance in barley by constitutive expression of heat and other abiotic stress-response genes. Temperature is one of the most crucial abiotic factors defining the yield potential of temperate cereal crops, such as barley. The regulators of heat shock response (HSR), heat stress transcription factors (Hsfs), modulate the transcription level of heat-responsive genes to protect the plants from heat stress. In this study, an Hsf from wheat (TaHsfA6b) is overexpressed in barley for providing thermotolerance. Transgenic barley lines overexpressing TaHsfA6b showed improvement in thermotolerance. The constitutive overexpression of a TaHsfA6b gene upregulated the expression of major heat shock proteins and other abiotic stress-responsive genes. RNA-seq and qRT-PCR analysis confirmed the upregulation of Hsps, chaperonins, DNAJ, LEA protein genes, and genes related to anti-oxidative enzymes in transgenic lines. Excessive generation and accumulation of reactive oxygen species (ROS) occurred in wild-type (WT) plants during heat stress; however, the transgenic lines reflected improved ROS homeostasis mechanisms, showing lesser ROS accumulation under high temperature. No negative phenotypic changes were observed in overexpression lines. These results suggest that TaHsfA6b is a regulator of HSR and its overexpression altered the expression patterns of some main stress-related genes and enhanced the thermotolerance of this cereal crop.

Genome-wide identification and expression analysis of Hsp70, Hsp90, and Hsp100 heat shock protein genes in barley under stress conditions and reproductive development.

Abiotic stress including extreme temperature disturbs the plant cellular homeostasis consequently limiting the yield potential of crop plants. Heat shock proteins (Hsps) are part of major rescue machinery of plants which aid to combat these stressed conditions by re-establishing protein homeostasis. Hsps with their chaperone and co-chaperone mechanisms regulate the activity of their substrate proteins in an ATP-dependent manner. In the present investigation, a genome-wide identification, evolutionary relationship, and comprehensive expression analysis of Hsp70, Hsp90, and Hsp100 gene families have been done in barley. The barley genome possesses 13 members of the Hsp70 gene family, along with 4 members of the Hsp110 subfamily, and 6 members of Hsp90 and 8 members of the Hsp100 gene family. Hsp genes are distributed on all 7 chromosomes of barley, and their encoded protein members are predicted to be localized to cell organelles such as cytosol, mitochondria, chloroplast, and ER. Despite a larger genome size, there are lesser members of these Hsp genes in barley, owing to less duplication events. The variable expression pattern obtained for genes encoding proteins localized to the same subcellular compartment suggests their diverse roles and involvement in different cellular responses. Expression profiling of these genes was performed by qRT-PCR in an array of 32 tissues, which showed a differential and tissue-specific expression of various members of Hsp gene families. We found the upregulation of HvHspc70-4, HvHsp70Mt70-2, HvHspc70-5a, HvHspc70-5b, HvHspc70-N1, HvHspc70-N2, HvHsp110-3, HvHsp90-1, HvHsp100-1, and HvHsp100-2 upon exposure to heat stress during reproductive development. Furthermore, their higher expression during heat stress, heavy metal stress, drought, and salinity stress was also observed in a tissue-specific manner.

Abscisic acid is a substrate of the ABC transporter encoded by the durable wheat disease resistance gene Lr34.

The wheat Lr34res allele, coding for an ATP-binding cassette transporter, confers durable resistance against multiple fungal pathogens. The Lr34sus allele, differing from Lr34res by two critical nucleotide polymorphisms, is found in susceptible wheat cultivars. Lr34res is functionally transferrable as a transgene into all major cereals, including rice, barley, maize, and sorghum. Here, we used transcriptomics, physiology, genetics, and in vitro and in vivo transport assays to study the molecular function of Lr34. We report that Lr34res results in a constitutive induction of transcripts reminiscent of an abscisic acid (ABA)-regulated response in transgenic rice. Lr34-expressing rice was altered in biological processes that are controlled by this phytohormone, including dehydration tolerance, transpiration and seedling growth. In planta seedling and in vitro yeast accumulation assays revealed that both LR34res and LR34sus act as ABA transporters. However, whereas the LR34res protein was detected in planta the LR34sus version was not, suggesting a post-transcriptional regulatory mechanism. Our results identify ABA as a substrate of the LR34 ABC transporter. We conclude that LR34res-mediated ABA redistribution has a major effect on the transcriptional response and physiology of Lr34res-expressing plants and that ABA is a candidate molecule that contributes to Lr34res-mediated disease resistance.

Drug-polymer miscibility, interactions, and precipitation inhibition studies for the development of amorphous solid dispersions for the poorly soluble anticancer drug flutamide.

The major goal of this research was to successfully formulate solid dispersion (SD) of the poorly soluble anticancer drug flutamide (FLT) using various hydrophilic polymers. Furthermore, to get more insight into SD, solid-state studies (miscibility and molecular interaction) were correlated with solution study (precipitation inhibition, dissolution). Hydrophilic polymers like PVP K90, HPMC, Eudragit EPO, and PEG 8000 were used at different drug-to-polymer w/w ratios. Solid-state miscibility studies were carried out using modulated differential scanning calorimetry (MDSC). SDs were prepared using solvent-evaporation technique and characterized by powder X-ray diffraction (PXRD) and MDSC. Infrared, Raman spectroscopy and molecular modeling were used to investigate drug-polymer interactions in the dispersions. Precipitation inhibition studies were carried out at various FLT-hydrophilic polymer ratios. Precipitation inhibition studies showed that PEG 8000 has the highest efficiency, followed by PVP K90, while HPMC and EPO showed no effect on precipitation inhibition. In the solid-state, MDSC of the physical mixture (PM) suggested that FLT is miscible to a greater extent with EPO and PEG 8000. Characterization of the amorphous dispersions using MDSC and PXRD concluded that FLT transformed from crystalline to amorphous form in the presence of PVP K90 and PEG 8000. Spectroscopic results confirmed stronger interaction of FLT with PVP K90 and PEG 8000, thereby confirming the in-solution precipitation and molecular modeling binding energy results. Amorphous dispersions formulated with PVP and PEG were stable and showed higher dissolution, an important property necessary to improve the physicochemical properties and drug delivery of poorly soluble anticancer drug FLT.

Drug-Lipid-Surfactant Miscibility for the Development of Solid Lipid Nanoparticles.

This research aimed to study the correlation between miscibility of flutamide (FLT), lipids and surfactant on the particle size of solid lipid nanoparticles (SLNs). Physical mixtures (PMs) of lipids-glyceryl monooleate (GMO), Precirol® (glyceryl palmitostearate, PRE), glyceryl monostearate (GMS), and Compritol® (glyceryl dibehenate, COM) were prepared with surfactant-Gelucire® (stearoyl polyoxyl-32 glycerides, GEL) 50/13 and 44/14. PMs were prepared in 5:2 w/w ratio (lipid:surfactant) and 2:1 w/w (Flutamide (FLT):lipids/GEL 50/13) by co-melting. Miscibility of PMs was investigated using modulated differential scanning calorimetry (MDSC). SLNs with and without drug were prepared using GEL 50/13 by the ultra-sonication method and particle size analysis was conducted. PMs of GMO, GMS, and PRE with both surfactants showed a decrease in the melting temperature, no change in melting and crystallization peak was observed with COM-GELs, indicating immiscibility. Similarly, MDSC data suggests good miscibility of FLT in GMO, GMS, and GEL 50/13 but not in PRE and COM. The particle size of drug-loaded SLNs prepared from GMO and GMS with GEL 50/13 was found to be 70.2 ± 5.4 and 92.6 ± 8.5 compared to > 200-nm particles obtained from PRE and COM. On lyophilization, an increase in particles size was observed with COM only. The particle size of SLNs with PRE and COM was prominently increased during stability studies indicating SLNs prepared with GMO and GMS are more stable due to miscibility and ability to reduce the crystallinity of FLT. The results established a good correlation between drug, lipids, and surfactants miscibility to the obtained particle size of SLNs before and after lyophilization. Graphical Abstract ᅟ.

Preparation, Characterization and Antioxidant Evaluation of Poorly Soluble Polyphenol-Loaded Nanoparticles for Cataract Treatment.

Cataract, one of the leading causes of blindness worldwide, is a condition in which complete or partial opacity develops in the lens of the eyes, thereby impairing vision. This study aimed to examine the potential therapeutic and protective effects of poorly soluble polyphenols like curcumin, resveratrol, and dibenzoylmethane, known to possess significant antioxidant activity. The polyphenols were loaded into novel lipid-cyclodextrin-based nanoparticles and characterized by particle size, polydispersity index, differential scanning calorimetry, thermogravimetric analysis, X-ray diffraction, scanning electron microscopy (SEM), entrapment efficiency, and release studies. Ferric-reducing ability of plasma and 2,2-diphenyl-1-picrylhydrazyl chemical assays were used to evaluate their antioxidant properties based on their free radical quenching ability. Biochemical in vitro assays were used to examine these polyphenols on hydrogen peroxide-induced formation of cataracts in bovine lenses by estimating total glutathione content and superoxide dismutase activity. Nanoparticles were thermostable and amorphous. Particle size of curcumin, resveratrol, and dibenzoylmethane nanoparticles were 331.0 ± 17.9 nm, 329.9 ± 1.9 nm, and 163.8 ± 3.2 nm, respectively. SEM confirmed porous morphology and XRD confirmed physical stability. Entrapment efficiency for curcumin-, resveratrol-, and dibenzoylmethane-loaded nanoparticles was calculated to be 84.4 ± 2.4%, 72.2 ± 1.5%, and 86.4 ± 0.6%, respectively. In vitro release studies showed an initial burst release followed by a continuous release of polyphenols from nanoparticles. Chemical assays confirmed the polyphenols' antioxidant activity. Superoxide dismutase and glutathione levels were found to be significantly increased (p < 0.05) after treatment with polyphenol-loaded nanoparticles than pure polyphenols; thus, an improved antioxidant activity translational into potential anticataract activity of the polyphenols when loaded into nanoparticles was observed as compared to pure polyphenols.

Pharmaceutical Topical Delivery of Poorly Soluble Polyphenols: Potential Role in Prevention and Treatment of Melanoma.

Melanoma is regarded as the fifth and sixth most common cancer in men and women, respectively, and it is estimated that one person dies from melanoma every hour in the USA. Unfortunately, the treatment of melanoma is difficult because of its aggressive metastasis and resistance to treatment. The treatment of melanoma continues to be a challenging issue due to the limitations of available treatments such as a low response rate, severe adverse reactions, and significant toxicity. Natural polyphenols have attracted considerable attention from the scientific community due to their chemopreventive and chemotherapeutic efficacy. It has been suggested that poorly soluble polyphenols such as curcumin, resveratrol, quercetin, coumarin, and epigallocatechin-3-gallate may have significant benefits in the treatment of melanoma due to their antioxidant, anti-inflammatory, antiproliferative, and chemoprotective efficacies. The major obstacles for the use of polyphenolic compounds are low stability and poor bioavailability. Numerous nanoformulations, including solid lipid nanoparticles, polymeric nanoparticles, micelles, and liposomes, have been formulated to enhance the bioavailability and stability, as well as the therapeutic efficacy of polyphenols. This review will provide an overview of poorly soluble polyphenols that have been reported to have antimetastatic efficacy in melanomas.

Preparation, Characterization, and In vitro Evaluation of Curcumin- and Resveratrol-Loaded Solid Lipid Nanoparticles.

Curcumin and resveratrol are natural compounds with significant anticancer activity; however, their bioavailability is limited due to poor solubility. This study aimed to overcome the solubility problem by means of solid lipid nanoparticles (SLN). 2-Hydroxypropyl ß-cyclodextrin (HPßCD) was selected from a range of polymers based on miscibility and molecular interactions. SLNs were obtained by probe sonication and freeze-drying curcumin-resveratrol with/without HPßCD incorporated in gelucire 50/13. SLNs were characterized by dynamic light scattering (DLS), zeta potential, powder X-ray diffractometry (PXRD), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), scanning electron microscopy (SEM), and physical stability. The in vitro release of drugs from the SLNs was performed by the direct dispersion method and analyzed using a validated UV-visible method. In vitro efficacy was tested using a colorectal cancer cell line. Curcumin-resveratrol-gelucire 50/13-HPßCD (CRG-CD) and curcumin-resveratrol-gelucire 50/13(CRG) SLNs showed a particle size from 100 to 150 nm and were not in the crystalline state per PXRD results. MDSC results complimented PXRD results by the absence of melting endotherm of curcumin; TGA showed no weight loss, confirming the absence of organic solvent residual, and the shape of the SLNs was confirmed as spherical by SEM. CRG SLNs were stable for 21 days with respect to particle size and zeta potential. MTT assay indicated better IC50 value for CRG as compared to CRG-CD. Hence, novel SLNs of curcumin and resveratrol incorporated in gelucire 50/13 and HPßCD were prepared and characterized to improve their bioavailability and anticancer activity.

Pathogen-inducible Ta-Lr34res expression in heterologous barley confers disease resistance without negative pleiotropic effects.

Plant diseases are a serious threat to crop production. The informed use of naturally occurring disease resistance in plant breeding can greatly contribute to sustainably reduce yield losses caused by plant pathogens. The Ta-Lr34res gene encodes an ABC transporter protein and confers partial, durable, and broad spectrum resistance against several fungal pathogens in wheat. Transgenic barley lines expressing Ta-Lr34res showed enhanced resistance against powdery mildew and leaf rust of barley. While Ta-Lr34res is only active at adult stage in wheat, Ta-Lr34res was found to be highly expressed already at the seedling stage in transgenic barley resulting in severe negative effects on growth. Here, we expressed Ta-Lr34res under the control of the pathogen-inducible Hv-Ger4c promoter in barley. Sixteen independent barley transformants showed strong resistance against leaf rust and powdery mildew. Infection assays and growth parameter measurements were performed under standard glasshouse and near-field conditions using a convertible glasshouse. Two Hv-Ger4c::Ta-Lr34res transgenic events were analysed in detail. Plants of one transformation event had similar grain production compared to wild-type under glasshouse and near-field conditions. Our results showed that negative effects caused by constitutive high expression of Ta-Lr34res driven by the endogenous wheat promoter in barley can be eliminated by inducible expression without compromising disease resistance. These data demonstrate that Ta-Lr34res is agronomically useful in barley. We conclude that the generation of a large number of transformants in different barley cultivars followed by early field testing will allow identifying barley lines suitable for breeding.

The wheat resistance gene Lr34 results in the constitutive induction of multiple defense pathways in transgenic barley.

The wheat gene Lr34 encodes an ABCG-type transporter which provides durable resistance against multiple pathogens. Lr34 is functional as a transgene in barley, but its mode of action has remained largely unknown both in wheat and barley. Here we studied gene expression in uninfected barley lines transgenic for Lr34. Genes from multiple defense pathways contributing to basal and inducible disease resistance were constitutively active in seedlings and mature leaves. In addition, the hormones jasmonic acid and salicylic acid were induced to high levels, and increased levels of lignin as well as hordatines were observed. These results demonstrate a strong, constitutive re-programming of metabolism by Lr34. The resistant Lr34 allele (Lr34res) encodes a protein that differs by two amino acid polymorphisms from the susceptible Lr34sus allele. The deletion of a single phenylalanine residue in Lr34sus was sufficient to induce the characteristic Lr34-based responses. Combination of Lr34res and Lr34sus in the same plant resulted in a reduction of Lr34res expression by 8- to 20-fold when the low-expressing Lr34res line BG8 was used as a parent. Crosses with the high-expressing Lr34res line BG9 resulted in an increase of Lr34sus expression by 13- to 16-fold in progenies that inherited both alleles. These results indicate an interaction of the two Lr34 alleles on the transcriptional level. Reduction of Lr34res expression in BG8 crosses reduced the negative pleiotropic effects of Lr34res on barley growth and vigor without compromising disease resistance, suggesting that transgenic combination of Lr34res and Lr34sus can result in agronomically useful resistance.

The wheat durable, multipathogen resistance gene Lr34 confers partial blast resistance in rice.

The wheat gene Lr34 confers durable and partial field resistance against the obligate biotrophic, pathogenic rust fungi and powdery mildew in adult wheat plants. The resistant Lr34 allele evolved after wheat domestication through two gain-of-function mutations in an ATP-binding cassette transporter gene. An Lr34-like fungal disease resistance with a similar broad-spectrum specificity and durability has not been described in other cereals. Here, we transformed the resistant Lr34 allele into the japonica rice cultivar Nipponbare. Transgenic rice plants expressing Lr34 showed increased resistance against multiple isolates of the hemibiotrophic pathogen Magnaporthe oryzae, the causal agent of rice blast disease. Host cell invasion during the biotrophic growth phase of rice blast was delayed in Lr34-expressing rice plants, resulting in smaller necrotic lesions on leaves. Lines with Lr34 also developed a typical, senescence-based leaf tip necrosis (LTN) phenotype. Development of LTN during early seedling growth had a negative impact on formation of axillary shoots and spikelets in some transgenic lines. One transgenic line developed LTN only at adult plant stage which was correlated with lower Lr34 expression levels at seedling stage. This line showed normal tiller formation and more importantly, disease resistance in this particular line was not compromised. Interestingly, Lr34 in rice is effective against a hemibiotrophic pathogen with a lifestyle and infection strategy that is different from obligate biotrophic rusts and mildew fungi. Lr34 might therefore be used as a source in rice breeding to improve broad-spectrum disease resistance against the most devastating fungal disease of rice.

Role of Molecular Interactions for Synergistic Precipitation Inhibition of Poorly Soluble Drug in Supersaturated Drug-Polymer-Polymer Ternary Solution.

We are reporting a synergistic effect of combined Eudragit E100 and PVP K90 in precipitation inhibition of indomethacin (IND) in solutions at low polymer concentration, a phenomenon that has significant implications on the usefulness of developing novel ternary solid dispersion of poorly soluble drugs. The IND supersaturation was created by cosolvent technique, and the precipitation studies were performed in the absence and the presence of individual and combined PVP K90 and Eudragit E100. The studies were also done with PEG 8000 as a noninteracting control polymer. A continuous UV recording of the IND absorption was used to observe changes in the drug concentration over time. The polymorphic form and morphology of precipitated IND were characterized by Raman spectroscopy and scanning electron microscopy. The change in the chemical shift in solution (1)H NMR was used as novel approach to probe IND-polymer interactions. Molecular modeling was used for calculating binding energy between IND-polymer as another indication of IND-polymer interaction. Spontaneous IND precipitation was observed in the absence of polymers. Eudragit E100 showed significant inhibitory effect on nuclei formation due to stronger interaction as reflected in higher binding energy and greater change in chemical shift by NMR. PVP K90 led to significant crystal growth inhibition due to adsorption on growing IND crystals as confirmed by modified crystal habit of precipitate in the presence of PVP K90. Combination of polymers resulted in a synergistic precipitation inhibition and extended supersaturation. The NMR confirmed interaction between IND-Eudragit E100 and IND-PVP K90 in solution. The combination of polymers showed similar peak shift albeit using lower polymer concentration indicating stronger interactions. The results established the significant synergistic precipitation inhibition effect upon combining Eudragit E100 and PVP K90 due to drug-polymer interaction.