Jelena Savić

Hairy root exudates of allelopathic weed Chenopodium murale L. induce oxidative stress and down-regulate core cell cycle genes in Arabidopsis and wheat seedlings

The effects of Chenopodium murale root exudates, applied as phytotoxic medias (PMs), were tested on Arabidopsis thaliana and Triticum aestivum. The effects of PMs, where wild-type roots (K), hairy roots derived from roots (R clones) or from cotyledons (C clones) were cultured, were different. K medium suppressed Arabidopsis germination, while other PMs reduced root and leaf elongation and the number of rosette leaves. R media were more phytotoxic than C media. Treatment of Arabidopsis with R8 down-regulated expression of core cell cycle genes: cyclin-dependent kinase (CDK) A1;1, four B-class CDKs, and cyclins CYCA3;1, CYCB2;4, CYCD4;2 and CYCH1 in root and shoot tips. Only CYCD2;1 transcript was elevated in treated shoots, but down-regulated in roots. Wheat Ta-CDC2 and Ta-CYCD2 genes showed the same expression profiles as their Arabidopsis counterparts, CDKA1;1 and CYCD2;1. PMs also caused increase of antioxidative enzyme activities in both plants. Exposure of Arabidopsis to PMs induced one catalase isoform, but repressed another, resulting in no net change of catalase activity. Wheat seedlings treated with PMs had catalase activity significantly elevated in all treatments, particularly in shoots. In both plants, PMs induced the activity of different peroxidase isozymes and total peroxidase activity. Both plants responded to phytotoxic treatments by induction of CuZn-superoxide dismutase. Thus, the phytotoxicity of C. murale root exudates is, at least partially, based on down-regulation of the cell cycle regulators and on generation of oxidative stress in the affected plants. We propose that C. murale root exudates should be considered as means of biological weed control.

Pyramiding rice cystatin OCI and OCII genes in transgenic potato (Solanum tuberosum L.) for resistance to Colorado potato beetle (Leptinotarsa decemlineata Say)

Proteinase inhibitors oryzacystatins I and II (OCI and OCII) have shown potential in controlling pests that utilize cysteine proteinases for protein digestion. In order to achieve an additive effect against Colorado potato beetle (CPB, Leptinotarsa decemlineata Say) digestive proteinases, we combined two individual OC genes in potato (Solanum tuberosum L.) cultivars Desiree, Dragačevka and Jelica. The OC genes were stacked into the potato genome by simultaneous co-transformation with both OCI and OCII genes or by sequential re-transformation of an OCI-expressing transgenic line with an OCII gene. Low constitutive and high wound-induced transcript levels of both OCI and OCII genes, directed by the inducible pin2 promoter, were detected in all doubly transformed lines from all three cultivars. Both recombinant OCs, detected by immunoblot, were in an active conformation as shown by their strong papain inhibitory activity. Although no significant differences in CPB larval mortality were observed when larvae fed on OCI/OCII transformed or control potato foliage, expression of both OCI and OCII in potato plants caused a reduction in CPB adult body weight and in weight of foliage consumed, indicating an additive effect. Overall, these results show that pyramiding of the OCI and OCII genes could be an effective strategy for breeding cultivars exhibiting elevated levels of resistance to CPB.

Docking Studies, Synthesis and Biological Evaluation of β-aryl-β-hydroxy propanoic Acids for Anti-inflammatory Activity.

BACKGROUND Nonsteriodal anti-inflammatory drugs (NSAIDs) are numerous and widely used for more than 60 years, but there is still a strong need for developing novel selective NSAIDs. The need is justified by the fact that nonselective NSAIDs can produce serious gastric side effects and that some of the selective NSAID are withdrawn due to their cardiotoxic side effects. METHODS Eight β-hydroxy-β-arylpropanoic acids, which belong to the arylpropanoic acid class of compounds, structurally similar to some nonsteroidal anti-inflammatory drugs (NSAIDs), were docked into 3D catalytic site of both cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2). Seven out of those eight acids were synthesized using already published modification of Reformatsky reaction additionally optimized by increasing temperature. Synthesized compounds were tested in vivo in order to elucidate anti-inflammatory activity, gastric tolerability and impact on liver function of rats. RESULTS Results of docking studies have indicated that all compounds have potential to selectively inhibit COX-2 isoform, but that the compounds containing polar substituents on phenyl ring are better inhibitors. Results of carrageenan-induced rat paw oedema test have shown that all compounds exhibit dose dependence and good gastric tolerability and none of the tested compounds have shown negative effect on liver function compared to ibuprofen. CONCLUSION The compound containing polar nitro group in para position has shown the best docking results, anti-inflammatory activity, low hepatotoxicity and good gastric tolerability.

Co‐expression of the proteinase inhibitors oryzacystatin I and oryzacystatin II in transgenic potato alters Colorado potato beetle larval development

Colorado potato beetle (CPB; Leptinotarsa decemlineata Say, Coleoptera: Chrysomelidae) has shown a remarkable adaptability to a variety of control measures. Although oryzacystatin I and II (OCI and OCII) have potential in controlling pests that use cysteine proteinases for food digestion, expression of a single OC gene in potato exhibited a minimal or no effect on CPB fitness traits. The aim of this study was to examine the effect of coexpressed OCI and OCII in potato (Solanum tuberosum L.) cultivars Desiree, Dragačevka and Jelica on CPB larvae. Growth parameters, consumption rates and food utilization, as well as activity of proteases of CPB larvae were assayed. Second and third instar larvae fed on transformed leaves molted earlier and had higher relative growth and consumption rates than larvae fed on nontransformed leaves, while efficiency of food utilization was unaffected. In contrast, fourth instar maximum weight gain and amount of leaves consumed were about 20% lower for the larvae fed on transgenic potato. Analysis of total protease activity of third instar larvae revealed reduction in overall proteolytic activity measured by azocasein hydrolysis, accompanied with inhibition of cysteine proteinase activity 24 h after ingestion of potato leaves expressing OCI and OCII. However, after long‐term feeding on transformed leaves proteolytic activities of larvae became similar to the controls. Although feeding on OCI/OCII leaves did not affect larval survival, coexpression of OC genes reduced the development time and thus significantly decreased plant damage caused by CPB larvae.

Docking Studies and α-Substitution Effects on the Anti-Inflammatory Activity of β-Hydroxy-β-arylpropanoic Acids

Six β-hydroxy-β-aryl propanoic acids were synthesised using a modification of Reformatsky reaction which has already been reported. These acids belong to the aryl propanoic acid class of compounds, structurally similar to the NSAIDs, such as ibuprofen, and an anti-inflammatory activity is thus expected. The aim of this work was to determine anti-inflammatory activity, examine gastric tolerability, and to carry out molecular docking experiments to identify potential COX-2 inhibitors among the β-hydroxy-β-aryl propanoic acids, and to elucidate the effect α-methyl substitution on the anti-inflammatory activity. Anti-inflammatory activity and gastric tolerability were determined on rats using carragenan induced paw oedema method, and docking studies were carried out using Autodock v4.0.1. The range of ED50 values is between 127 µmol/kg and 15 µmol/kg, while the result for ibuprofen is 51.7 µmol/kg. Only slight hyperaemia or few petechiae were spotted on rat’s stomach. The results indicate that all compounds possess significant anti-inflammatory activity after oral administration, and that 2-methyl-3-hydroxy-3,3-diphenyl-propanoic acid has greatest activity, surpassing that of ibuprofen, a standard NSAID. Another compound, 3-hydroxy-3,3-diphenylpropanoic acid, shows activity matching that of ibuprofen, and is non-chiral and is proven to be non-toxic. The most of investigated compounds have interactions with P3 anchor site like COX-2 selective inhibitors. No tested substances or ibuprofen produced any significant gastric lesions.

Extraordinary Adaptive Plasticity of Colorado Potato Beetle: “Ten-Striped Spearman” in the Era of Biotechnological Warfare

Expanding from remote areas of Mexico to a worldwide scale, the ten-striped insect, the Colorado potato beetle (CPB, Leptinotarsa decemlineata Say), has risen from being an innocuous beetle to a prominent global pest. A diverse life cycle, phenotypic plasticity, adaptation to adverse conditions, and capability to detoxify or tolerate toxins make this insect appear to be virtually “indestructible”. With increasing advances in molecular biology, tools of biotechnological warfare were deployed to combat CPB. In the last three decades, genetically modified potato has created a new challenge for the beetle. After reviewing hundreds of scientific papers dealing with CPB control, it became clear that even biotechnological means of control, if used alone, would not defeat the Colorado potato beetle. This control measure once again appears to be provoking the potato beetle to exhibit its remarkable adaptability. Nonetheless, the potential for adaptation to these techniques has increased our knowledge of this pest and thus opened possibilities for devising more sustainable CPB management programs.

In vitro prediction of gastrointestinal absorption of novel β-hydroxy-β-arylalkanoic acids using PAMPA technique

&NA; Prediction of gastrointestinal absorption of thirteen newly synthesized &bgr;‐hydroxy‐&bgr;‐arylalkanoic acids (HAA) and ibuprofen was performed using PAMPA test. The highest values of PAMPA parameters (%T and Papp) were calculated for 1C, 1B and 2C and these parameters were significantly lower in comparison to ibuprofen. QSPR analysis was performed in order to identify molecular descriptors with the highest influence on %T and − logPapp and to create models which could be used for the design of novel HAA with improved gastrointestinal absorption. Obtained results indicate that introduction of branched side chain, as well as introduction of substituents on one phenyl ring (which disturb symmetry of the molecule) could have positive impact on gastrointestinal absorption. On the basis of these results, six novel HAA were designed and PAMPA parameters %T and − logPapp were predicted by use of selected QSPR models. Designed derivatives should have better gastrointestinal absorption than HAA tested in this study. Graphical abstract Figure. No caption available.

Physiological and cell ultrastructure disturbances in wheat seedlings generated by Chenopodium murale hairy root exudate

Chenopodium murale L. is an invasive weed species significantly interfering with wheat crop. However, the complete nature of its allelopathic influence on crops is not yet fully understood. In the present study, the focus is made on establishing the relation between plant morphophysiological changes and oxidative stress, induced by allelopathic extract. Phytotoxic medium of C. murale hairy root clone R5 reduced the germination rate (24% less than control value) of wheat cv. Nataša seeds, as well as seedling growth, diminishing shoot and root length significantly, decreased total chlorophyll content, and induced abnormal root gravitropism. The R5 treatment caused cellular structural abnormalities, reflecting on the root and leaf cell shape and organization. These abnormalities mostly included the increased number of mitochondria and reorganization of the vacuolar compartment, changes in nucleus shape, and chloroplast organization and distribution. The most significant structural changes were observed in cell wall in the form of amoeboid protrusions and folds leading to its irregular shape. These structural alterations were accompanied by an oxidative stress in tissues of treated wheat seedlings, reflected as increased level of H2O2 and other ROS molecules, an increase of radical scavenging capacity and total phenolic content. Accordingly, the retardation of wheat seedling growth by C. murale allelochemicals may represent a consequence of complex activity involving both cell structure alteration and physiological processes.

Application of biopartitioning micellar chromatography and QSRR modeling for prediction of gastrointestinal absorption and design of novel β-hydroxy-β-arylalkanoic acids

&NA; Gastrointestinal absorption of thirteen novel &bgr;‐hydroxy‐&bgr;‐arylalkanoic acids (HAA) with anti‐inflammatory activity was predicted by use of biopartitioning micellar chromatography and compared to ibuprofen. All tested HAA have lower retention factors (k) and lower expected gastrointestinal absorption than ibuprofen, whereas derivatives with the highest values of k are 1C, 2APTF and 2C. Quantitative structure‐retention relationship (QSRR) analysis was performed in order to identify molecular descriptors with the highest influence on k and ANN(k) model was selected as optimal. Descriptors which form this model (nBM, P_VSA_LogP_8 and Eta_L) indicate that replacement of phenyl ring with a saturated or partially unsaturated one, as well as presence of halogens and nitro group should positively affect k values. On the basis of these conclusions, six novel HAA were designed and selected QSRR model was used for the prediction of their k values. Graphical abstract Figure. No caption available.

Insect resistance to sugar beet pests mediated by a Beta vulgaris proteinase inhibitor transgene.

We transformed sugar beet (Beta vulgaris) hairy roots and Nicotiana benthamiana plants with a Beta vulgaris root gene (BvSTI) that codes for a serine proteinase inhibitor. BvSTI is a root gene cloned from the F1016 breeding line that has moderate levels of resistance to the sugar beet root maggot (Tetanops myopaeformis), a major pest of sugar beet. Root maggot damage deforms roots and predisposes them to other pests and pathogens that reduce yield and quality. Transformants had high levels of BvSTI or GUS gene expression driven by the constitutive 35S or the BvSTI gene specific promoter, respectively. Polyacrylamide gel electrophoresis zymograms revealed clear zones that corresponded to proteinase inhibitor (PI) activities at ~24, 26 and 28 kDa in the sugar beet BvSTI transformants and 26 and 28 kDa in the N. benthamiana plants. Several insect pests of sugar beet and tobacco were bioassayed for resistance on the BvSTI-transgenic plant materials. Spodoptera frugiperda (fall armyworm), Spodoptera exigua (beet armyworm) and Manduca sexta (tobacco hornworm) larvae that were fed tobacco leaves or sugar beet roots that express the BvSTI gene exhibited higher mortality rates or were delayed in growth and development relative to control larvae. Since serine proteases comprise the major digestive enzymes in root maggot midguts, our findings suggest that the BvSTI gene is likely involved in root maggot resistance mechanisms in the F1016 genotype.