Screening, isolation, and molecular characterization of putative fragrance-related transcripts from Vanda Mimi Palmer

Abstract

The aims of this study were to isolate and characterize putative fragrance-related cDNAs from the floral cDNA library of Vanda Mimi Palmer, an orchid hybrid that has won several international awards for its sweet fragrance. A total of 1,000,000 pfu were screened by hybridizing cDNA library plaques with fully open flower cDNA probe of Vanda Mimi Palmer representing all mRNAs expressed during daytime. The clones that gave positive signals were in vivo excised and PCR-amplified inserts were subjected to reverse-Northern analysis by hybridizing with cDNA probes of fully open flower of Vanda Mimi Palmer (fragrant orchid) and its bud or fully open flower of Vanda Tan Chay Yan (non-fragrant orchid) separately. The clones up-regulated in fully open flower stage of Vanda Mimi Palmer compared to its bud stage or fully open flower of Vanda Tan Chay Yan were sequenced. Sequence analyses showed the presence of eight putative fragrance-related cDNAs of which two were putative Vanda Mimi Palmer 4-(cytidine 5 0 -diphospho)-2-Cmethyl-D-erythritol kinase (VMPCMEK) and Vanda MimiPalmer cytochrome P450 protein (VMPCyP450). These two transcripts were selected for full-length cDNA isolation and expression analysis by real-time RT-PCR. The VMPCMEK transcript encodes a polypeptide of 400 amino acid residues, while the VMPCyP450 encodes 538 amino acid residues. Relative expression analysis of VMPCMEK and VMPCyP450 transcripts by real-time RT-PCR showed up-regulated expressions in floral tissues compared to vegetative tissues, and both were found to be developmentally regulated.

Floral micromorphology and transcriptome analyses of a fragrant Vandaceous Orchid, Vanda Mimi Palmer, for its fragrance production sites

“…The immature stage of VMP’s bud as previously reported [ 5 ] did not emit detectable fragrance compound until it achieved its fully-open flower stage. Such similar expression profiles were also obtained from our previous studies for phenylacetaldehyde synthase (VMPPAAS), sesquiterpene synthase (VMPSTS) and alcohol acyltransferase (VMPAAT) [ 5 , 15 , 16 ]. This observation also concurred with our findings of the high distribution of trichomes found at the edges of the petals and sepals, which might reflect their involvement in fragrance biosynthesis and emission.…”

“…Both fully-opened flower and bud had well-developed glandular trichomes, respectively, but only fully-opened flower produced strong fragrance; hence, we postulated that the immaturity of the trichomes could be a factor in explaining why the bud was scentless. In our molecular findings, some of the fragrance-related transcripts were detected in the bud but preferentially at higher level in blooming flowers [ 13 , 16 ]. There was no significant difference ( p > 0.05) in terms of trichome density per 5 mm 2 coverage area between petals and sepals (dorsal and lateral) from different developmental stages of the flower (Table 1 ).…”

Molecular Cloning, Sequencing, and Characterization of a Putative Acetyl-CoA-C-acetyltransferase cDNA from a Highly Fragrant Orchid Hybrid Vanda Mimi Palmer

“…Among these, more than 120 genera are found naturally in Peninsular Malaysia [1,2]. Vanda Mimi Palmer, a hybrid of Vanda Tan Chay Yan and Vanda tessellata (Roxb.)…”

“…Over the years, the numbers of fragrance-related cDNAs that have been isolated and characterized particularly in [18], V. Mimi Palmer 4-(cytidine 5 -diphospho)-2-C-methyld-erythritol kinase (VMPCMEK) and V. Mimi Palmer cytochrome P450 (VMPCyP450) [1]. Recently, we reported the successful expression and functional characterization of the VMPSTS in Lactococcus lactis [19].…”