Ercan Arican

Small RNA and degradome deep sequencing reveals drought-and tissue-specific micrornas and their important roles in drought-sensitive and drought-tolerant tomato genotypes

Summary Drought stress has adverse impacts on plant production and productivity. MicroRNAs (miRNAs) are one class of noncoding RNAs regulating gene expression post‐transcriptionally. In this study, we employed small RNA and degradome sequencing to systematically investigate the tissue‐specific miRNAs responsible to drought stress, which are understudied in tomato. For this purpose, root and upground tissues of two different drought‐responsive tomato genotypes (Lycopersicon esculentum as sensitive and L. esculentum var. cerasiforme as tolerant) were subjected to stress with 5% polyethylene glycol for 7 days. A total of 699 conserved miRNAs belonging to 578 families were determined and 688 miRNAs were significantly differentially expressed between different treatments, tissues and genotypes. Using degradome sequencing, 44 target genes were identified associated with 36 miRNA families. Drought‐related miRNAs and their targets were enriched functionally by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. Totally, 53 miRNAs targeted 23 key drought stress‐ and tissue development‐related genes, including DRP (dehydration‐responsive protein), GTs (glycosyltransferases), ERF (ethylene responsive factor), PSII (photosystem II) protein, HD‐ZIP (homeodomain‐leucine zipper), MYB and NAC‐domain transcription factors. miR160, miR165, miR166, miR171, miR398, miR408, miR827, miR9472, miR9476 and miR9552 were the key miRNAs functioning in regulation of these genes and involving in tomato response to drought stress. Additionally, plant hormone signal transduction pathway genes were differentially regulated by miR169, miR172, miR393, miR5641, miR5658 and miR7997 in both tissues of both sensitive and tolerant genotypes. These results provide new insight into the regulatory role of miRNAs in drought response with plant hormone signal transduction and drought‐tolerant tomato breeding.

Reduced Polyphenol Oxidase Activity in Transgenic Potato Plants Associated with Reduced Wound-Inducible Browning Phenotypes

ABSTRACT In potato the polyphenol oxidase (PPO) mediated wound-inducible browning phenotypes is one of the major drawback for food industry. In order to investigate and reduce PPOs expression for browning we obtained the potato specific PPO cDNAs using Vicia faba L. specific (Locus VVU 83274) primers and cloned them under control of the cauliflower mosaic virus 35S promoter. Transgenic potato plants (Solanum tuberosum L.) that show down expression of PPOs were obtained using Agrobacterium mediated transformation. Wounded transgenic plants with reduced PPO activity exhibited a great amount of reduced browning when compared with their control plants. Our results confirm the importance of PPO mediated phenolic oxidation in browning discoloration of potato suggesting the possibility of manipulation of PPO expression

Inhibition of crown-gall tumorigenesis with plant extracts

Measurement of the inhibition of crown-gall tumors on potato discs is an antitumor bioassay method for detection of anti-tumor compounds from higher plants. In this study, after surface sterilization, tuber discs were co-cultivated with Agrobacterium tumefaciens B6S3 for two days. The discs were then inoculated on MS media with the crude extracts obtained from different parts of various plants – Allium sativum L. (Liliaceae), Salvia verbenaca L., Rosmarinus officinalis L., Ocimum basiculum L., Lavandula stoechas L. (Lamiaceae), Althaea cannabina L. (Malvaceae), Petroselinum sativum Mill. (Apiaceae), Pelargonium radicula L. (Geraniaceaea), Juglans regia L. (Juglandaceae), Platanus orientalis L. (Platanaceae), Laurus nobilis L. (Lauraceae), Ranunculus ficaria L. (Ranunculaceae), and Abies equi-trojani Aschers. et Sint. Ex Boiss (Pinaceae). Tumors appeared after 10-15 days, and tumor inhibition was detected only in six plant extracts of 13 (A. sativum, R. officinalis, P. orientalis, L. nobilis, R. ficaria, and A. equi-trojani) when compared to control material. Constitutive tumor inhibition activities were higher in these plant extracts (63.5%, 56.1%, 61.7%, 54.6%, 69.7%, and 57.9% at 0 min; 58.8%, 54.6%, 58.8%, 48.4%, 62.6%, and 51.6% for 15 min after bacterial inoculation).

EVALUATION OF THE APOPTOTIC AND ANTIPROLIFERATIVE ACTIVITIES OF PACLITAXEL IN EHRLICH ASCITES TUMOR CELLS

ABSTRACT Taxol (paclitaxel, PAC) is a diterpene with antitumor activity against a variety of human neoplasms. PAC cytotoxicity is thought to derive mainly from a stabilization of microtubules as a result of enhanced tubulin polymerization that leads to an accumulation of cells in G2/M phase of cell cycle. In this study, the cytotoxic effect occurred by PAC in tumor cells in vitro were examined. Mouse mammary gland carcinoma-derived Ehrlich Ascites Tumor Cells (EATC) were employed as tumor cell line. Results of the experiments were evaluated with cell kinetic parameters such as growth rate, mitotic index, apoptotic index and DNA fragmentation. IC30 and IC50 concentrations of PAC (6 μg/ml and 12 μg/ml, respectively) were applied to the cell line for 0–24 hours. The proliferation rate of EATC was inhibited by PAC concentrations compared to control with increasing treatment time (2–24 hours). The inhibition of growth rate was higher in 12 μg/ml PAC treatment than those in 6 μg/ml treatment. In our studies, when the mitotic index parameter data was evaluated to determine which phase of the cell cycle was affected by PAC to cause the repression of cell reproduction, it was found that PAC exposes its cytotoxic effect by causing cell accumulation at mitosis. The accumulation of the cells resulted in an increase in mitotic index values, which was an expected consequence of PAC treatment. In addition to the effect of PAC on mitotic cell accumulation, the results on its effect on apoptotic index parameter support the findings about its repressive function on cell reproduction and its effect on mitotic index increase. It was observed that depending on the drug treatments, increase of mitotic index, inhibition of growth rate and apoptotic index in EATC were increased with respect to control, being of statistically significant (p<0.01-p<0.001). And the DNA fragmentation levels of EATC induced by PAC increased significantly.

Amplification of Specific Genes by using RT-PCR Technique in Plants

ABSTRACT Reverse transcription polymerase chain reaction (RT-PCR) is a sensitive and powerful method for analyzing RNA. In this study, genes encoding polyphenol oxidase and glucanase enzymes were amplified by using RT-PCR technique in potato and chickpea, respectively. It was also determined that both of the genes were members of multigene family.

Effects of Naphthalene Acetic Acid on Transformation Frequency of Potato and Tobacco Via Agrobacterium Rhizogenes

ABSTRACTTransformations of potato (Solanum tuberosum cv. Yayla kizi) and tobacco (Nicotiana tabacum cv. Samsun NN) were achieved using tuber discs and internodes via Agrobacterium rhizogenes 8196 wild strain on MS (11) media supplemented with 5 μM Naphthalene Acetic Acid (NAA). In this study, we examined the effects of NAA on hairy root formation of potato and tobacco. Cultures of bacteria were concentrated at O.D. 600=0.2 for inoculation and MS media supplemented with 1 μM Cefotaxime (Cx) and 5 μM NAA were used for co-cultivation. Transformed hairy roots were obtained after three weeks in a regulated plant growth cabinet at 25 °C with 16 hours light followed by 8 hours dark period. DNA was extracted from these roots and digested using EcoR I and Hind III restriction endonucleases, and transformation was confirmed using the Southern blot hybridization method with [γ32P] CTP labeled Ri plasmid DNA of A. rhizogenes 8196.

Transformation of Potato (Solanum Tuberosum L.) using Tuber Discs and Stem Explants

ABSTRACTTransformation of three potato cultivars (Desiree, Isola and Anac) was achieved using tuber discs and callus tissue derived from cultured stems via co-cultivation with Agrobacterium tumafaciens Ti plasmid based vectors. Neomycin phosphotransferase (NPT II) marker and β-glucuronidase (GUS) reporter genes were used in transfer-optimization studies. First selection of transgenics was done on selective Murashige and Skoog (MS) media containing 100 mg/l kanamycin and 500 mg/l cefotaxime. Histochemical assays were done for GUS activity using X-Gluc substrat. In addition polymerase chain reaction based Southern blot analyses revealed the existence of the transferred genes. The highest transformation frequency (10%) was obtained with cultivar Desiree in MS media supplied with 5 mg/l zeatin riboside and 1.5 mg/l indolacetic acid. However, Anac gave the lowest frequency using tuber discs (3%) and stem explants (2%). This protocol could be useful for the improvement of potato through gene manipulation.

Elicitation triterpene yield in Alstonia scholaris cell cultures via synergetic organisms

ABSTRACT Cell cultures of Alstonia scholaris were treated with homogenates of Candida albicans, Fusarium oxysporum, Penicillium avelanium and Saccharomyces cerevisiae. The impact caused by the concentration, exposure time and the type of synergetic organisms on the accumulation of pentacyclic triterpenes was observed. When exposed to biotic elicitors for longer periods, some cell lines doubled the production of those triterpenes. S. cerevisiae homogenate was the best elicitor of triterpenes in all cell lines investigated.

Effects of Hypobaric Conditions on Apoptosis Signalling Pathways in HeLa Cells

Nowadays increasing effectiveness in cancer therapy and investigation of formation of new strategies that enhance antiproliferative activity against target organs has become a subject of interest. Although the molecular mechanisms of apoptosis can not be fully explained, it is known that cell suicide program existing in their memory genetically is activated by pathophysiological conditions and events such as oxidative stress. Low pressure (hypobaric) conditions that create hypoxia promote apoptosis by inhibiting cell cycling. In this study, determination of the effects of fractional hypobaric applications at different times on HeLa cells at cellular and molecular levels were targeted. Experiments were carried out under hypobaric conditions (35.2 kPa) in a specially designed hypobaric cabin including 2% O2 and 98% N. Application of fractional hypobaric conditions was repeated two times for 3 hours with an interval of 24 hours. At the end of the implementation period cells were allowed to incubate for 24 hours for activation of repair mechanisms. Cell kinetic parameters such as growth rate (MTT) and apoptotic index were used in determination of the effect of hypobaric conditions on HeLa cells. Also in our study expression levels of the Bcl-2 gene family that have regulatory roles in apoptosis were determined by the RT-PCR technique to evaluate molecular mechanisms. The results showed that antiproliferative effect of hypobaric conditions on HeLa cells started three hours from the time of application and increased depending on the period of exposure. While there was a significant decrease in growth rate values, there was a significant increase in apoptotic index values (p<0.01). Also molecular studies showed that hypobaric conditions caused a significant increase in expression level of proapoptotic gene Bax and significant decrease in antiapoptotic Bfl-1. Consequently fractional application of hypobaric conditions on HeLa cell cultures increased both antiproliferative and apoptotic effects and these effects were triggered by the Bax gene.

Transgenic Investigation of Canola (Brassica napus L.)

Investigating transgenic characteristics of Californium, Jura, Elvis and Orkan varieties of canola hasbeen carried out in this study. These plants are widely cultivated in Turkey. The Canola varieties weregrown in an established tissue cultu re and the total DNA isolated.The 35S and pNOS promoter region of the transgene were scanned using classic and multiplex PCR techniques. The transfer of transgenic characteristic to the NAD gene region confirms the accuracy of the PCR technique. This study, has determined that Californium, Jura, Elvis and Orkan varieties of canola consist of transgenic characteristics and all the four varieties have multiple insertion region in terms of CaMV 35S and Pnos promoters.