Diterpene Resin Acids and Olefins in Calabrian Pine (Pinus nigra subsp. laricio (Poiret) Maire) Oleoresin: GC-MS Profiling of Major Diterpenoids in Different Plant Organs, Molecular Identification and Expression Analysis of Diterpene Synthase Genes

Abstract

A quali-quantitative analysis of diterpenoid composition in tissues obtained from different organs of Pinus nigra subsp. laricio (Poiret) Maire (Calabrian pine) was carried out. Diterpene resin acids were the most abundant diterpenoids across all the examined tissues. The same nine diterpene resin acids were always found, with the abietane type prevailing on the pimarane type, although their quantitative distribution was found to be remarkably tissue-specific. The scrutiny of the available literature revealed species specificity as well. A phylogeny-based approach allowed us to isolate four cDNAs coding for diterpene synthases in Calabrian pine, each of which belonging to one of the four groups into which the d3 clade of the plants’ terpene synthases family can be divided. The deduced amino acid sequences allowed predicting that both monofunctional and bifunctional diterpene synthases are involved in the biosynthesis of diterpene resin acids in Calabrian pine. Transcript profiling revealed differential expression across the different tissues and was found to be consistent with the corresponding diterpenoid profiles. The isolation of the complete genomic sequences and the determination of their exon/intron structures allowed us to place the diterpene synthase genes from Calabrian pine on the background of current ideas on the functional evolution of diterpene synthases in Gymnosperms.

Monoterpene Synthase Genes and Monoterpene Profiles in Pinus nigra subsp. laricio

“…Taken together, the above results suggested that the quantitatively profile of monoterpenes in P. laricio is remarkably tissue-specific, and the perusal of the available literature indicated a species specificity as well; thus, confirming our previous results on the diterpenoids from the same conifer species [ 28 ]. As it was shown by several studies [ 10 , 30 , 31 ], there is a notable variation in the monoterpene composition between different genera and species of conifer trees.…”

“…Another aspect of potential interest in the non-model conifer species studied here, in view of its possible physiological, ecological, and technological implications, concerns the reciprocal quantitative relationships among the major terpenoids components of oleoresin, as well as the developmental and environmental factors driving the differential distribution of them in plant tissues. In our aforementioned twin study [ 28 ], we analysed quantitatively, in the identical plant material studied here and by adopting an essentially similar GC–MS approach, the diterpenoid components of oleoresin in the form of diterpene resin acids (DRAs), i.e., the stable and functional conformers deriving from the combined action of DTPSs and of cytochrome P450 monooxygenases [ 1 ]. By comparing the results obtained in the two experiments ( Figure 1 and [ 28 ]) on a DW basis, total monoterpenes as a whole in P. laricio are about six times more abundant than total DRAs.…”

“…The isolation of P. laricio MTPS genes allowed a tissue-specific gene expression analysis to compare with the corresponding GC–MS monoterpene profiles. The results presented here on P. laricio are compared to, and discussed against, those obtained in the aforementioned twin study of ours [ 28 ], focusing on the diterpenoid components of oleoresin from the same species, in an attempt to expand and consolidate the basic knowledge on this non-model conifer, possibly leading to applications of interest in bio-based technology.…”

“…It became apparent from the above-mentioned work that most of the existing knowledge concerning the genetics and metabolism of monoterpenes in conifers was obtained from model Pinaceae species, for which large transcriptomic and genomic resources are available. In previous works of ours [ 6 , 27 , 28 ], we began to gain insight into the ecological and functional roles of the terpenes produced by the non-model conifer Pinus nigra J.F.Arnold subsp. laricio Palib.…”

“…In one of our previous works on P. laricio [ 28 ], five different plant tissues were analysed in terms of the oleoresin diterpenoids composition, and, in parallel, the isolation, characterization, and expression analyses of diterpene synthase (DTPS) genes were carried out in the same plant material. To broaden the characterization of the terpene oleoresins in this non-model conifer species, the same combined biochemical and genetic approach is adopted in the present work, in which, again, for the first time to the best of our knowledge, we use gas chromatography–mass spectrometry (GC–MS) to determine the monoterpenes profiles in five different tissues of P. laricio .…”