Cloning and Functional Analysis of NtHDR in ‘Jinzhanyintai’ of Narcissus tazetta var. chinensis M.Roem

Abstract

The gene encoding 1-hydroxy-2-methyl-2-(E)-butenyl-4-pyrophosphate reductase, a key enzyme at the end of the methylerythritol 4-phosphate pathway (MEP), was cloned from the petals of Narcissus tazetta var. Chinensis ‘Jinzhanyintai’ and named NtHDR (accession number OQ739816). NtHDR exhibits a full-length coding sequence spanning 1380 base pairs, encoding a total of 460 amino acids. Through phylogenetic-tree analysis, it has been determined that NtHDR shared the closest evolutionary relationship with monocotyledons, specifically Asparagus officinalis and Zingiber officinale. Utilizing quantitative real-time PCR analysis, it was observed that NtHDR exhibited significant expression in both petals and corona, with expression levels varying throughout the flowering process of Narcissus. Specifically, in petals, NtHDR expression demonstrated a pattern of initial increase followed by subsequent decrease, while in corona, it consistently increased. Subsequent subcellular-localization experiments indicated that NtHDR was localized within the chloroplasts. To investigate the functional impact of NtHDR, a stable transformation was performed, where NtHDR was introduced into Nicotiana benthamiana. The resulting transgenic N. benthamiana flowers were analyzed using solid-phase microextraction coupled with gas chromatography–mass spectrometry (SPME-GC-MS) to characterize their volatile components. The analysis revealed the presence of the monoterpene compound linalool, as well as the phenylpropanoids benzyl alcohol and phenylethanol, within the floral fragrance components of the transgenic N. benthamiana plants. However, these compounds were absent in the floral fragrance components of wild-type tobacco plants, thus highlighting the impact of NtHDR transformation on the floral scent profile.