Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated9 (Cas9) endonuclease system is a powerful RNA-guided genome editing tool. CRISPR/Cas9 has been well studied in model plant species for targeted genome editing. However, few studies have been reported on plant species without whole genome sequence information. Currently, no study has been performed to manipulate metabolic pathways using CRISPR/Cas9. In this study, the type II CRISPR/SpCas9 system was used to knock out, via nonhomologous end-joining genome repair, the 4′OMT2 in opium poppy (Papaver somniferum L.), a gene which regulates the biosythesis of benzylisoquinoline alkaloids (BIAs). For sgRNA transcription, viral-based TRV and synthetic binary plasmids were designed and delivered into plant cells with a Cas9 encoding-synthetic vector by Agrobacterium-mediated transformation. InDels formed by CRISPR/Cas9 were detected by sequence analysis. Our results showed that the biosynthesis of BIAs (e.g. morphine, thebaine) was significantly reduced in the transgenic plants suggesting that 4′OMT2 was efficiently knocked-out by our CRISPR-Cas9 genome editing approach. In addition, a novel uncharacterized alkaloid was observed only in CRISPR/Cas9 edited plants. Thus, the applicabilitiy of the CRISPR/Cas9 system was demonstrated for the first time for medicinal aromatic plants by sgRNAs transcribed from both synthetic and viral vectors to regulate BIA metabolism and biosynthesis.
Plants are frequently exposed to microorganisms like fungi, bacteria, and viruses that cause biotic stresses. Fusarium head blight (FHB) is an economically risky wheat disease, which occurs upon Fusarium graminearum (Fg) infection. Moderately susceptible (cv. “Mizrak 98”) and susceptible (cv. “Gun 91”) winter type bread wheat cultivars were subjected to transcriptional profiling after exposure to Fg infection. To examine the early response to the pathogen in wheat, we measured gene expression alterations in mock and pathogen inoculated root crown of moderately susceptible (MS) and susceptible cultivars at 12 hours after inoculation (hai) using 12X135K microarray chip. The transcriptome analyses revealed that out of 39,179 transcripts, 3668 genes in microarray were significantly regulated at least in one time comparison. The majority of differentially regulated transcripts were associated with disease response and the gene expression mechanism. When the cultivars were compared, a number of transcripts and expression alterations varied within the cultivars. Especially membrane related transcripts were detected as differentially expressed. Moreover, diverse transcription factors showed significant fold change values among the cultivars. This study presented new insights to understand the early response of selected cultivars to the Fg at 12 hai. Through the KEGG analysis, we observed that the most altered transcripts were associated with starch and sucrose metabolism and gluconeogenesis pathways.
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.
Papaver bracteatum Lindl., P. orientale L., and P. pseudo-orientale (Fedde) Medw., species of section Oxytona Bernh. in the genus Papaver L., are found widely in the native flora of Turkey. They are commercially important for their high alkaloid content. Because genus Papaver section Oxytona has similar morphological characters, it is not easy to distinguish these species. We used the inter-simple sequence repeat (ISSR) molecular marker system to determine the molecular characterisation of this section. In this study, 20 ISSR primers have been studied in 180 accessions collected from 5 different regions of Turkey. A total of 82 bands were obtained, of which 80 were polymorphic. Average genetic distance was found to be 0.35, the Shannon index was 0.50, and the polymorphism rate was 96.97%. The primer AT10 had the smallest number of bands, which was 3, while the primers AT19 and AT3 had the greatest number of bands, which was 5. In conclusion, the ISSR marker system can be used for the classification of section Oxytona.
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