• Journal of Applied Biological Chemistry
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• v.43 no.4
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• pp.230-234
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• 2000

A new method to assay the capsaicinoid synthetase (CS) activity was developed by utilizing NADHcoupled enzyme systems involving pyruvate kinase and lactate dehydrogenase. CS activities in Capsicum placenta, depending upon the kinetics of the NADH oxidation, revealed almost the same profile as compared with those shown using an HPLC-based method. When the substrates, 8-methyl nonanoic acid and vanillylamine, for the CS enzyme were employed separately or simultaneously, it appeared that the two-step reaction, acyl-CoA formation and condensation with vanillyla~ne, of the CS enzyme was a coupled reaction. Thus, this assay method of the CS enzyme can be considered as an alternative to the HPLC-based method, since it has the advantages of rapidity and simplicity as well as reliability when compared with the existing method.

• Journal of Applied Biological Chemistry
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• v.43 no.3
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• pp.152-155
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• 2000

The contents of various capsaicinoids viz. nordihydrocapsaicin (NDC), capsaicin (CAP), and dihydrocapsaicin (DHC) were determined in different parts of fruits (placenta, pericarps, seeds, and whole fruits) at different developmental stages after flowering and compared with the capsaicinoid synthetase (CS) activity. The capsaicinoid contents were very low up to 24 days after flowering (DAF), and there was a significant increase at 36 DAF in all parts of fruits. The enzyme activity of placenta increased to maximum at 24 DAF, and thereafter it gradually decreased. There were no significant amounts of enzyme activities in other parts of the fruits. In Subicho (inbred line) the content of DHC was slightly higher than CAP in all parts. of the fruits throughout the development stages of fruits, whereas in Chung Yang the CAP content was higher compared to the DHC content. The contents of total capsaicinoids in Chung Yang were also higher than Subicho. However, the crude enzyme extract obtained from Chung Yang led to the synthesis of DHC almost exclusively when the substrate, 8-methyl nonanoic acid, was added to the reaction mixture. Our results suggest that the composition of individual analogue of capsaicinoids depends upon the substrate available in the fruits.

• Molecules and Cells
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• v.19 no.2
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• pp.262-267
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• 2005

The capsaicinoid synthetase (CS) gene cosegregated perfectly with the C locus, which controls the presence of pungency, in 121 $F_2$ individuals from a cross between 'ECW123R' and 'CM334', both of Capsicum annuum. We concluded that CS and C are tightly linked. Sequence analysis of the genes of four pungent and four non-pungent pepper lines showed that the non-pungent peppers had a 2,529 bp-deletion in the 5' upstream region of CS. We have developed molecular markers of the C locus to detect pungency at the seedling stage. Based on the deleted sequence, we developed five SCAR markers, two of them being codominant. These SCAR markers will be useful for easy, accurate, and early detection of non-pungent individuals in breeding programs.

• Plant Biotechnology Reports
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• v.5 no.2
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• pp.157-167
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• 2011

In order to establish a reliable and highly efficient method for genetic transformation of pepper, a monitoring system featuring GFP (green fluorescent protein) as a report marker was applied to Agrobacteriummediated transformation. A callus-induced transformation (CIT) system was used to transform the GFP gene. GFP expression was observed in all tissues of $T_0$, $T_1$ and $T_2$ peppers, constituting the first instance in which the whole pepper plant has exhibited GFP fluorescence. A total of 38 T0 peppers were obtained from 4,200 explants. The transformation rate ranged from 0.47 to 1.83% depending on the genotype, which was higher than that obtained by CIT without the GFP monitoring system. This technique could enhance selection power by monitoring GFP expression at the early stage of callus in vitro. The detection of GFP expression in the callus led to successful identification of the shoot that contained the transgene. Thus, this technique saved lots of time and money for conducting the genetic transformation process of pepper. In addition, a co-transformation technique was applied to the target transgene, CaCS (encoding capsaicinoid synthetase of Capsicum) along with GFP. Paprika varieties were transformed by the CaCS::GFP construct, and GFP expression in callus tissues of paprika was monitored to select the right transformant.