Physiological and oxidative stress response of carrot (Daucus carota L.) to jumping plant-louse Bactericera trigonica Hodkinson (Hemiptera: Psylloidea) infestation.

BACKGROUND: Carrot is an important vegetable crop grown worldwide. The major economic problem in carrot cultivation is yellow disease caused by Bactericera trigonica, which induces biotic stress and has the greatest impact on crop productivity. Comprehensive studies on the mechanism of carrot defense response to biotic stress caused by B. trigonica infestation have yet to be conducted. METHODS: The changes in photosynthetic pigments, proline, TPC, H2O2 and MDA content, DPPH radical scavenging ability, and antioxidant enzyme activity of SOD, CAT, and POX in carrot leaves in response to insect sex (female and male), rapid response (during the first six hours), and long-term response to B. trigonica infestation were evaluated. RESULTS: The results of our study strongly suggest that B. trigonica infestation causes significant changes in primary and secondary metabolism and oxidative status of carrot leaves. Photosynthetic pigment content, TPC, and DPPH and CAT activities were significantly reduced in carrot leaves in response to insect infestation. On the other hand, proline, H2O2 content, and the activity of the antioxidant enzymes superoxide dismutase and peroxidase were increased in carrot leaves after B. trigonica infestation. The results indicate that B. trigonica attenuates and delays the oxidative stress responses of carrot, allowing long-term feeding without visible changes in the plant. Carrot responded to long-term B. trigonica infestation with an increase in SOD and POX activity, suggesting that these enzymes may play a key role in plant defense mechanisms. CONCLUSIONS: This is the first comprehensive study strongly suggesting that B. trigonica infestation causes significant changes in primary and secondary metabolism and an attenuated ROS defense response in carrot leaves that enables long-term insect feeding. The information provides new insights into the mechanisms of carrot protection against B. trigonica infestation.

Foliar application of methyl jasmonate affects impatiens walleriana growth and leaf physiology under drought stress.

In this study, the effects of foliar applied methyl jasmonate (MeJA) on drought-stressed Impatiens walleriana growth and leaf physiology parameters: stomatal conductance, chlorophyll, flavonoid, anthocyanin, and nitrogen balance index (NBI), were evaluated. These parameters could serve as indicators of drought tolerance of I. walleriana, a popular horticultural plant worldwide that is very sensitive to drought. The experiment included four treatments: control, drought-stressed plants sprayed with distilled water, drought-stressed plants sprayed with 5 µM MeJA, and drought-stressed plants sprayed with 50 µM MeJA. Foliar spraying with MeJA was performed twice: seven days before and on the day of drought induction. The stressed plant groups were non-irrigated to achieve soil water contents (SWC) of 15 and 5%, while control plants were well-watered throughout the experiment (35-37% SWC). The results of this study showed that drought significantly reduced I. walleriana fresh and dry shoot weight, as well as total leaf area, but did not impact on dry matter content. The foliar application of MeJA improved growth parameters of I. walleriana, depending on the elicitor concentration and drought intensity. Stomatal conductance was slightly reduced at 5% SWC, and foliar applied MeJA at both concentrations. The flavonoid index was slightly reduced at 15 and 5% SWC when 50 µM MeJA was foliar applied, while there were no observed changes in the anthocyanin index in any treatments. The foliar application of 50 µM MeJA increased the chlorophyll index and NBI of I. walleriana at 5% SWC, indicating a contribution of the elicitor to plant drought tolerance at the physiological level.

Alterations in Physiological, Biochemical, and Molecular Responses of Impatiens walleriana to Drought by Methyl Jasmonate Foliar Application.

Drought stress affects plant growth and development through several mechanisms, including the induction of oxidative stress. To cope with drought, plants have drought tolerance mechanisms at the physiological, biochemical, and molecular levels. In this study, the effects of foliar application of distilled water and methyl jasmonate (MeJA) (5 and 50 µM) on the physiological, biochemical, and molecular responses of Impatiens walleriana during two drought regimes (15 and 5% soil water content, SWC) were investigated. The results showed that plant response depended on the concentration of the elicitor and the stress intensity. The highest chlorophyll and carotenoid contents were observed at 5% SWC in plants pre-treated with 50 µM MeJA, while the MeJA did not have a significant effect on the chlorophyll a/b ratio in drought-stressed plants. Drought-induced formation of hydrogen peroxide and malondialdehyde in plants sprayed with distilled water was significantly reduced in plant leaves pretreated with MeJA. The lower total polyphenol content and antioxidant activity of secondary metabolites in MeJA-pretreated plants were observed. The foliar application of MeJA affected the proline content and antioxidant enzyme activities (superoxide dismutase, peroxidase, and catalase) in plants that suffered from drought. The expression of abscisic acid (ABA) metabolic genes (IwNCED4, IwAAO2, and IwABA8ox3) was the most affected in plants sprayed with 50 µM MeJA, while of the four analyzed aquaporin genes (IwPIP1;4, IwPIP2;2, IwPIP2;7, and IwTIP4;1), the expression of IwPIP1;4 and IwPIP2;7 was strongly induced in drought-stressed plants pre-treated with 50 µM MeJA. The study's findings demonstrated the significance of MeJA in regulating the gene expression of the ABA metabolic pathway and aquaporins, as well as the considerable alterations in oxidative stress responses of drought-stressed I. walleriana foliar sprayed with MeJA. The results improved our understanding of this horticulture plant's stress physiology and the field of plant hormones' interaction network in general.

Role of Sodium Nitroprusside on Potential Mitigation of Salt Stress in Centaury (Centaurium erythraea Rafn) Shoots Grown In Vitro.

Soil salinity is one of the most common abiotic stressors that affects plant growth and development. The aim of this work was to investigate the influence of sodium nitroprusside (SNP), a donor of nitric oxide (NO), on the physiological response of common centaury (Centaurium erythraea) shoots grown under stress conditions caused by sodium chloride (NaCl) in vitro. Centaury shoots were first grown on nutrient medium containing different SNP concentrations (50, 100 and 250 µM) during the pretreatment phase. After three weeks, the shoots were transferred to nutrient media supplemented with NaCl (150 mM) and/or SNP (50, 100 or 250 µM) for one week. The results showed that salinity decreased photosynthetic pigments, total phenolic content and DPPH (1,1-diphenyl-2-picrylhydrazyl radical) concentration. The activities of antioxidant enzymes, namely superoxide dismutase (SOD), catalase (CAT) and peroxidase (POX), were also reduced under salt stress. However, MDA concentration was decreased, while H2O2 and proline content did not drastically change under the stress conditions caused by NaCl. Exogenous application of SNP altered the biochemical parameters of centaury shoots grown under salt stress. In this case, increased photosynthetic pigment content, total phenolics and proline content were noted, with reduced MDA, but not H2O2, concentration was observed. In addition, the exogenous application of SNP increased the degree of DPPH reduction as well as SOD, CAT and POX activities.

Physiological Response, Oxidative Stress Assessment and Aquaporin Genes Expression of Cherry Tomato (Solanum lycopersicum L.) Exposed to Hyper-Harmonized Fullerene Water Complex.

The rapid production and numerous applications of nanomaterials warrant the necessity and importance of examining nanoparticles in terms to their environmental and biological effects and implications. In this study, the effects of a water-soluble hyper-harmonized hydroxyl-modified fullerene (3HFWC) on cherry tomato seed germination, seedlings growth, physiological response and fruiting was evaluated. Changes in the photosynthetic pigments content, oxidative stress assessment, and aquaporin genes expression in cherry tomato plants were studied after during short- and long-term continuous exposure to 3HFWC nanosubstance (200 mg/L). Increased levels of photosynthetic pigments in leaves, lycopene in fruits, decreased levels of hydrogen peroxide content, activation of cellular antioxidant enzymes such as superoxide dismutase, catalase and peroxidase and increased aquaporin gene expression (PIP1;3, PIP1;5 and PIP2;4) were observed in 3HFWC nanosubstance-exposed plants in comparison to control, untreated cherry tomato plants. The 3HFWC nanosubstance showed positive effects on cherry tomato seed germination, plantlet growth and lycopene content in fruits and may be considered as a promising nanofertilizer.

Association between microRNAs 10b/21/34a and acute toxicity in glioblastoma patients treated with radiotherapy and temozolomide.

A personalized approach to chemoradiation is important in reducing its potential side effects and identifying a group of patients prone to toxicity. MicroRNAs have been shown to have a predictive potential for radiotoxicity. The goal of the study was to test if levels of miRNA in peripheral blood mononuclear cells of glioblastoma patients are associated with toxicity and to identify the peak time point for toxicity. MicroRNA-10b/21/34a levels were measured in 43 patients with and without toxicity, at baseline, at the 15th, and at the 30th fraction by Real-Time quantitative Polymerase Chain Reaction. MicroRNA-10b/21 levels increased with toxicity grade (p = 0.014; p = 0.013); miR-21/34a levels were significantly different between patients with and without toxicity at the 15th fraction (p = 0.030; p = 0.045), while miR-34a levels significantly changed during treatment (p < 0.001). All three miRNAs showed a significantly high positive correlation with one another. MiR-34a might be considered as a predictive factor for toxicity due to its changes during treatment, and differences between the groups with and without toxicity; miR-10b might be used to predict toxicity; miR-10b/21 might be used for predicting the grade of toxicity in GB patients.

Phytohormone profiles in non-transformed and AtCKX transgenic centaury (Centaurium erythraea Rafn) shoots and roots in response to salinity stress in vitro.

Plant hormones regulate numerous developmental and physiological processes. Abiotic stresses considerably affect production and distribution of phytohormones as the stress signal triggers. The homeostasis of plant hormones is controlled by their de novo synthesis and catabolism. The aim of this work was to analyse the contents of total and individual groups of endogenous cytokinins (CKs) as well as indole-3-acetic acid (IAA) in AtCKX overexpressing centaury plants grown in vitro on graded NaCl concentrations (0, 50, 100, 150, 200 mM). The levels of endogenous stress hormones including abscisic acid (ABA), salicylic acid (SA) and jasmonic acid (JA) were also detected. The elevated contents of total CKs were found in all analysed centaury shoots. Furthermore, increased amounts of all five CK groups, as well as enhanced total CKs were revealed on graded NaCl concentrations in non-transformed and AtCKX roots. All analysed AtCKX centaury lines exhibited decreased amounts of endogenous IAA in shoots and roots. Consequently, the IAA/bioactive CK forms ratios showed a significant variation in the shoots and roots of all AtCKX lines. In shoots and roots of both non-transformed and AtCKX transgenic centaury plants, salinity was associated with an increase of ABA and JA and a decrease of SA content.

Molecular Characterization and Expression of Four Aquaporin Genes in Impatiens walleriana During Drought Stress and Recovery.

Aquaporins comprise a large group of transmembrane proteins responsible for water transport, which is crucial for plant survival under stress conditions. Despite the vital role of aquaporins, nothing is known about this protein family in Impatiens walleriana, a commercially important horticultural plant, which is sensitive to drought stress. In the present study, attention is given to the molecular characterization of aquaporins in I. walleriana and their expression during drought stress and recovery. We identified four I. walleriana aquaporins: IwPIP1;4, IwPIP2;2, IwPIP2;7 and IwTIP4;1. All of them had conserved NPA motifs (Asparagine-Proline-Alanine), transmembrane helices (TMh), pore characteristics, stereochemical properties and tetrameric structure of holoprotein. Drought stress and recovery treatment affected the aquaporins expression in I. walleriana leaves, which was up- or downregulated depending on stress intensity. Expression of IwPIP2;7 was the most affected of all analyzed I. walleriana aquaporins. At 15% and 5% soil moisture and recovery from 15% and 5% soil moisture, IwPIP2;7 expression significantly decreased and increased, respectively. Aquaporins IwPIP1;4 and IwTIP4;1 had lower expression in comparison to IwPIP2;7, with moderate expression changes in response to drought and recovery, while IwPIP2;2 expression was of significance only in recovered plants. Insight into the molecular structure of I. walleriana aquaporins expanded knowledge about plant aquaporins, while its expression during drought and recovery contributed to I. walleriana drought tolerance mechanisms and re-acclimation.

Morpho-Physiological and Molecular Evaluation of Drought and Recovery in Impatiens walleriana Grown Ex Vitro.

This study was carried out to examine the drought effect on development, physiological, biochemical and molecular parameters in Impatiens walleriana grown ex vitro. Experiment design included three treatments: Control plants-grown under optimal watering (35%-37% of soil moisture content), drought-stressed plants-non-irrigated to reach 15% and 5% of soil moisture content and recovery plants-rehydrated for four days to reach optimal soil moisture content. Drought reduced fresh weight, total leaf area, as well as dry weight of I. walleriana shoots. Drought up-regulated expression of abscisic acid (ABA) biosynthesis genes 9-cis-epoxycarotenoid dioxygenase 4 (NCED4) and abscisic aldehyde oxidase 2 (AAO2) and catabolic gene ABA 8'-hydroxylase 3 (ABA8ox3) which was followed by increased ABA content in the leaves. Decrement in water potential of shoots during the drought was not accompanied with increased amino acid proline content. We detected an increase in chlorophyll, carotenoid, total polyphenols and flavonols content under drought conditions, as well as malondialdehyde, hydrogen peroxide and DPPH (1,1'-diphenyl-2-picrylhydrazyl) activity. Increased antioxidant enzyme activities (superoxide dismutase, peroxidase and catalase) throughout drought were also determined. Recovery treatment was significant for neutralizing drought effect on growth parameters, shoot water potential, proline content and genes expression.

Breaking the Dormancy of Snake's Head Fritillary (Fritillaria meleagris L.) In Vitro Bulbs-Part 2: Effect of GA3 Soaking and Chilling on Sugar Status in Sprouted Bulbs.

The bulb is the main propagation organ of snake's head fritillary (Fritillaria meleagris L.), a horticulturally attractive and rare geophyte plant species. In this study, we investigated the effect of soaking bulbs in GA3 solution (1, 2, and 3 mg L-1) combined with low-temperature treatment (7 °C) on breaking the dormancy of in vitro bulbs. Sugar status (total soluble sugars, glucose, and fructose content) was analyzed in different parts of the sprouted bulbs. The results showed that the soluble sugar concentration was highest in bulbs soaked in GA3. The main sugar in fritillary bulbs was glucose, while fructose content was much lower. Glucose concentration dramatically increased after bulb chilling (7 °C), and its accumulation was predominantly detected in the lower sprout portion during the first weeks of sprouting. Sugar concentration was significantly lower in nonchilled bulbs, which indicates the importance of low temperature in bulb development and sprouting.

Effects of Exogenous Salicylic Acid on Drought Response and Characterization of Dehydrins in Impatiens walleriana.

Impatiens walleriana is a valued ornamental plant sensitive to drought stress. We investigated whether the foliar application of 2mM salicylic acid (SA) can protect potted I. walleriana plants from drought stress. The plants were divided into: watered plants, drought-stressed plants, watered plants treated with SA and drought-stressed plants treated with SA. The number of flowers and flower buds, relative water content (RWC), contents of malondialdehyde (MDA) and proline (Pro) and the activities of superoxide dismutases, catalases and peroxidases were recorded at different time points. Three dehydrin sequences were identified in de novo assembled leaf transcriptome: IwDhn1, IwDhn2.1 and IwDhn2.2. Drought stress caused wilting, floral abortion, reduction of RWC and increased MDA-an indicator of lipid peroxidation. In response to drought, Impatiens accumulated Pro and induced chloroplastic Cu/ZnSOD and two peroxidase isoforms. The most remarkable drought response was strong induction of IwDhn2.1 and IwDhn2.2. Rehydration restored RWC, Pro level, Cu/ZnSOD activity and dehydrins expression in drought-stressed plants approximately to the values of watered plants.SA had ameliorating effects on plants exposed to drought, including prevention of wilting, preservation of RWC, increased Pro accumulation, modulation of antioxidative activities and remarkable decrease of lipid peroxidation, but without effects on flowers' preservation.

Breaking the Dormancy of Snake's Head Fritillary (Fritillaria meleagris L.) In Vitro Bulbs-Part 1: Effect of GA3, GA Inhibitors and Temperature on Fresh Weight, Sprouting and Sugar Content.

Bulbs are the main vegetative reproductive organs of Fritillaria meleagris L. In nature, as well as in vitro, they become dormant and require low temperatures for further growth during the next vegetative period. In the present study, using 10 µM of gibberellic acid (GA3), or gibberellin biosynthesis (GA) inhibitors-ancymidol (A) and paclobutrazol (P)-the dynamic changes in soluble sugars, fructose and glucose content, fresh weight and sprouting capacity were investigated. F. meleagris bulbs were cultured on medium with GA3 and GA inhibitors for 1, 2 and 5 weeks at two different temperatures (24 and 7 °C). GA3 improved bulb fresh weight, as well as sprouting percentage at both tested temperatures, compared to the control. The highest fresh weight increase (57.7%) and sprouting rate (29.02%) were achieved when bulbs were grown at 24 °C for 5 weeks. In addition, soluble sugar content was the highest in bulbs grown for 5 weeks on medium supplemented with GA3. The main sugar in fritillary bulbs was glucose, while fructose content was lower. The sensitivity of bulbs to GA inhibitors differed and significantly affected sugar content in bulbs. To our knowledge, this is the first study of the sugar composition in F. meleagris bulbs during breaking of the bulb's dormancy and its sprouting.

A Survival Analysis with Identification of Prognostic Factors in a Series of 110 Patients with Newly Diagnosed Glioblastoma Before and After Introduction of the Stupp Regimen: A Single-Center Observational Study.

BACKGROUND: Current treatment protocol for glioblastoma multiforme (GBM) is based on maximal safe resection followed by the Stupp protocol. In Serbia, temozolomide was introduced as adjuvant therapy in 2011. The aims of this study were to confirm the safety and efficacy on overall and progression-free survival of the Stupp protocol and evaluate the influence of prognostic factors in one of the largest series of patients with GBM treated over a 2-year period. METHODS: Between January 2010 and December 2012, 110 patients with newly diagnosed GBM underwent surgical removal at the Neurooncology Department of the Clinic Center of Serbia. Patients were divided into 2 groups according to postoperative treatment. Group A (n = 24 patients), treated before January 2011, received adjuvant standard radiation therapy and carmustine (bis-chloroethyl-nitrosourea), and group B (n = 86 patients), treated after January 2011, received postoperative treatment according to the Stupp protocol. RESULTS: The Stupp protocol had a significant favorable impact on overall survival at 1-year follow-up (79.1% in group B vs. 62.5% in group A; P = 0.016); no differences were noted in regard to progression-free survival. Multivariate analysis identified younger age and gross total resection of tumor as positive prognostic factors. CONCLUSIONS: Adoption of the Stupp protocol had a favorable impact on overall, but not on progression-free, survival rate. Wider surgical resection involving the peritumoral brain zone, as confirmed by univariate and multivariate analysis, represents the most favorable prognostic factor.

A rare case of isolated adrenal metastasis of invasive ductal breast carcinoma.

INTRODUCTION: Isolated adrenal metastases of invasive ductal breast carcinoma are extremely rare. We report a case with isolated left adrenal metastases, verified three years after diagnosed breast carcinoma. CASE OUTLINE: A 58-year-old female patient with a right breast tumor, clinically staged as IIIA (T2N2M0) started neoadjuvant anthracycline chemotherapy after biopsy which revealed invasive ductal breast carcinoma. Immunohistochemical findings of tumor biopsy showed hormonal steroid receptors for estrogen and progesterone negative, and human epidermal growth factor receptor 2 (HER2) positive. After 4 cycles of chemotherapy and partial tumor regression the patient underwent radical mastectomy. Definite histopathological analysis confirmed the diagnosis of invasive ductal carcinoma. The patient continued treatment with adjuvant chemotherapy to cumulative dose of anthracyclines, postoperative radiotherapy and adjuvant trastuzumab for one year. Three years later abdominal computerized tomography showed tumor in the left adrenal gland as the only metastatic site. Left adrenalectomy was performed and histopathological finding confirmed breast cancer metastases. Postoperatively, the patient received 6 cycles of docetaxel with trastuzumab and continued trastuzumab until disease progression. One year after left adrenalectomy control abdominal computerized tomography showed a right adrenal tumor with retroperitoneal lymphadenopathy. Treatment with capecitabine was continued for one year, but eventually she developed brain metastasis causing lethal outcome. CONCLUSION: In order to better understand metastatic pathways of invasive ductal breast carcinoma, publications of individual patient cases diagnosed with rare metastatic sites should be encouraged. This might improve our understanding of metastatic behavior of breast cancer and stimulate further clinical research.

Elimination of TSWV from impatiens hawkerii Bull. and regeneration of virus-free plant

The possibility for obtaining virus free plants from Impatiens hawkerii Bull. shoots infected with Tomato spotted wilt virus (TSWV) through meristem-tip culture was examined. TSWV presence in I. hawkerii plants was detected by DAS-ELISA and RT-PCR and identification of the virus was confirmed by sequencing one of the chosen isolate (GenBank Accesion CQ132190). Meristem-tip explants (0.3-1.5 mm) from virus-infected shoots are cultured on MS media supplemented with different concentrations of the cytokinins, CPPU or TDZ (0.01-1.0 uM), respectively. Using this system, a large number of in vitro shoots could be produced from a single explant. Also, cytokinins showed a stimulatory effect on the length, fresh and dry weights of the newly formed shoots. Plant pigments content in I. hawkerii shoots increased significantly in the presence of cytokinins. Rooting of shoots was spontaneous on the same media. Rooted plantlets were transferred to soil where 97 percent successfully acclimatized. By DAS-ELISA and RT-PCR, 80 percent of the in vitro plantlets were shown to be a virus-free. Considering these, the present protocol seems to be an efficient method for in vitro generation of virus-free I. hawkerii plantlets by meristem tip cultures.