Sebahattin Özcan

Regulation of the alkaloid biosynthesis by miRNA in opium poppy

Opium poppy (Papaver somniferum) is an important medicinal plant producing benzylisoquinoline alkaloids (BIA). MicroRNAs (miRNAs) are endogenous small RNAs (sRNAs) of approximately 21 nucleotides. They are noncoding, but regulate gene expression in eukaryotes. Although many studies have been conducted on the identification and functions of plant miRNA, scarce researches on miRNA regulation of alkaloid biosynthesis have been reported. In this study, a total of 316 conserved and 11 novel miRNAs were identified in opium poppy using second-generation sequencing and direct cloning. Tissue-specific regulation of miRNA expression was comparatively analysed by miRNA microarray assays. A total of 232 miRNAs were found to be differentially expressed among four tissues. Likewise, 1469 target transcripts were detected using in silico and experimental approaches. The Kyoto Encyclopedia of Genes and Genomes pathway analyses indicated that miRNA putatively regulates carbohydrate metabolism and genetic-information processing. Additionally, miRNA target transcripts were mostly involved in response to stress against various factors and secondary-metabolite biosynthesis processes. Target transcript identification analyses revealed that some of the miRNAs might be involved in BIA biosynthesis, such as pso-miR13, pso-miR2161 and pso-miR408. Additionally, three putatively mature miRNA sequences were predicted to be targeting BIA-biosynthesis genes.

Differential competence for in vitro adventitous rooting of grass pea (Lathyrus sativus L.)

Grass pea (Lathyrus sativus L.) family leguminosae is cultivated as an important food and feed crop all over the world. It is very recalcitrant and difficult to regenerate and root under in vitro conditions. In this cotext, the study was carried out in three steps to find out the effects of three auxins [naphthalene acetic acid, indole 3 butyric acid, indole-3-acetic acid (IAA)], four sucrose concentrations and six types of substrate most suited for plant growth and helpful in acclimatisation of grass pea. The results showed that 2xa0mgxa0L−1 IAA, 3xa0% sucrose was most suitable for rooting of grass pea. When different concentrations of sucrose were supplied to optimum concentration of IAA in Murashige and Skoog medium, 4.5xa0% sucrose concentration induced maximum number of 13.70 roots per explants that had positive impact on root length, fresh and dry weight of roots, plant height and morphology of the growing plants. There was 92.66xa0% acclimatisation and survival rate of these plants using peat moss compared to five other substrates used in this study. The developing plants were vigorous, flowered and set seed contained in pods under glass house conditions. It is concluded that rooting is affected by type and concentration of plant growth regulators and type of substrate has direct bearing on acclimatisation, flowering, pod and seed set of grass pea. As such this paper reports an efficient rooting and acclimatisation system of grass pea that will be very useful in future genetic transformation and breeding for improved characteristics.

Functional Characterization of 4'OMT and 7OMT Genes in BIA Biosynthesis.

Alkaloids are diverse group of secondary metabolites generally found in plants. Opium poppy (Papaver somniferum L.), the only commercial source of morphinan alkaloids, has been used as a medicinal plant since ancient times. It produces benzylisoquinoline alkaloids (BIA) including the narcotic analgesic morphine, the muscle relaxant papaverine, and the anti-cancer agent noscapine. Though BIAs play crucial roles in many biological mechanisms their steps in biosynthesis and the responsible genes remain to be revealed. In this study, expressions of 3-hydroxy-N-methylcoclaurine 4′–methyltransferase (4′OMT) and reticuline 7-O-methyltransferase (7OMT) genes were subjected to manipulation to functionally characterize their roles in BIA biosynthesis. Measurements of alkaloid accumulation were performed in leaf, stem, and capsule tissues accordingly. Suppression of 4′OMT expression caused reduction in the total alkaloid content in stem tissue whereas total alkaloid content was significantly induced in the capsule. Silencing of the 7OMT gene also caused repression in total alkaloid content in the stem. On the other hand, over-expression of 4′OMT and 7OMT resulted in higher morphine accumulation in the stem but suppressed amount in the capsule. Moreover, differential expression in several BIA synthesis genes (CNMT, TYDC, 6OMT, SAT, COR, 4′OMT, and 7OMT) were observed upon manipulation of 4′OMT and 7OMT expression. Upon silencing and overexpression applications, tissue specific effects of these genes were identified. Manipulation of 4′OMT and 7OMT genes caused differentiated accumulation of BIAs including morphine and noscapine in capsule and stem tissues.

Powdery mildews observed on Onobrychis spp. in Turkey

Erysiphe trifolii and Leveillula taurica on Onobrychis spp. in Turkey, are described and illustrated.

Expression of GNA and biting site-restricted cry1Ac in cotton; an efficient attribution to insect pest management strategies

Insect-resistant transgenic cotton has been commercialized for two decades. Most of the introduced cultivars express Bt gene(s) constitutively under the control of 35S promoter in whole-plant tissues. However, there have been other promoters considered by researchers to confine the toxin expression to targeted organ and tissues. We developed a triple-gene construct including GNA, cry1Ac and cp4 epsps genes. We attempted to confine cry1Ac expression to insect biting sites by cloning it to downstream of a wound-inducible promoter isolated from Asparagus officinalis (AoPR1). Moreover, to broaden the range of resistance, GNA was driven by the 35S promoter to target the sap-sucking insects like aphids which impose large losses in cotton production. To select the transformants in selection medium and for glyphosate tolerance, GNA and cry1Ac genes were accompanied with cp4 epsps gene. Two binary vectors harboring desired genes were constructed and utilized in the study (pGTGNAoC1AC and pGTGN35C1AC). Transformation of cultivar GSN-12 was carried out by employing Agrobacterium tumefaciens strain EHA105. Plantlets were primarily screened under glyphosate (N-phosphonomethyl glycine) selection pressure and subsequently subjected to molecular and biotoxicity assays. Introduction of cry1Ac and GNA to cotton plant conferred resistance to Spodoptera littoralis and Aphis gossypii Glover. Restriction of cry1Ac toxin protein to insect biting sites along with a plant lectin attributes significantly to insect pest management strategies.