Abstract
Pine oleoresin is a major source of terpenes, consisting of turpentine (mono- and sesquiterpenes) and rosin (diterpenes) fractions. Higher oleoresin yields are of economic interest, since oleoresin derivatives make up a valuable source of materials for chemical industries. Oleoresin can be extracted from living trees, often by the bark streak method, in which bark removal is done periodically, followed by application of stimulant paste containing sulfuric acid and other chemicals on the freshly wounded exposed surface. To better understand the molecular basis of chemically-stimulated and wound induced oleoresin production, we evaluated the stability of 11 putative reference genes for the purpose of normalization in studying Pinus elliottii gene expression during oleoresinosis. Samples for RNA extraction were collected from field-grown adult trees under tapping operations using stimulant pastes with different compositions and at various time points after paste application. Statistical methods established by geNorm, NormFinder, and BestKeeper softwares were consistent in pointing as adequate reference genes HISTO3 and UBI. To confirm expression stability of the candidate reference genes, expression profiles of putative P. elliottii orthologs of resin biosynthesis-related genes encoding Pinus contorta β-pinene synthase [PcTPS-(−)β-pin1], P. contorta levopimaradiene/abietadiene synthase (PcLAS1), Pinus taeda α-pinene synthase [PtTPS-(+)αpin], and P. taeda α-farnesene synthase (PtαFS) were examined following stimulant paste application. Increased oleoresin yields observed in stimulated treatments using phytohormone-based pastes were consistent with higher expression of pinene synthases. Overall, the expression of all genes examined matched the expected profiles of oleoresin-related transcript changes reported for previously examined conifers.
Highlights
Pine oleoresin is a major source of terpenes, consisting of turpentine and rosin fractions
The use of inaccurate reference genes can lead to inconsistent results, when variations in the rate of transcription are small between sample groups (Dheda et al, 2005; Etschmann et al, 2006)
Eleven putative reference genes were selected for expression studies on P. elliottii oleoresinosis based on previous reports that indicated them as suitable genes for normalization of expression profiles in different species and in relation to other biological processes (Gonçalves et al, 2005; Wang et al, 2010; de VegaBartol et al, 2013)
Summary
Pine oleoresin is a major source of terpenes, consisting of turpentine (mono- and sesquiterpenes) and rosin (diterpenes) fractions. Oleoresin represents a key element of defense in conifers and its production can be affected by biotic and abiotic factors, which include mechanical injury, pathogen attack, water availability, seasonality, and chemical stimulating treatments (Rodrigues-Corrêa and Fett-Neto, 2012). Oleoresin is extracted for commercial purposes from living trees, often by the bark streak method, in which bark removal is done periodically, followed by application of chemical stimulant paste containing sulfuric acid and other chemicals on the freshly wounded exposed surface (Rodrigues-Corrêa et al, 2013). Chemical stimulant pastes may contain phytohormones or their precursors (e.g., ethylene, auxin, salicylic acid), metal ions (enzyme activators or co-factors, such as potassium and iron), or reactive oxygen species generators (e.g., paraquat), all of which increase resin production and/or facilitate its flow by activating defense responses (RodriguesCorrêa and Fett-Neto, 2012). Terpenes are derived from isopentenyl pyrophosphate (IPP), produced either through the mevalonate pathway in the cytosol-endoplasmic reticulum or by deoxi-xylulose-5-phosphate pathway in the plastids (Phillips and Croteau, 1999)
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