Validation of endogenous reference genes for RT-qPCR analysis in adult slash pine
under oleoresinosis: effect of stimulant pastes on monoterpene biosynthesis
Fernanda de Costa1, Magnus Riffel Kerber1, Júlio César de Lima2, Thanise Nogueira
Füller1, Kelly Cristine da Silva Rodrigues3 and Arthur Germano Fett-Neto1,4
1
Plant Physiology Laboratory, Department of Botany, Federal University of Rio Grande
do Sul (UFRGS), Porto Alegre, RS, Brazil
2
Molecular Genetics Laboratory, University of Passo Fundo, Passo Fundo, RS, Brazil
3
Regional Integrated University of Alto Uruguai and Missões (URI-FW), Frederico
Westphalen, RS, Brazil
4
Center for Biotechnology, UFRGS, Porto Alegre, RS, Brazil
Oleoresinosis is a defense mechanism of conifers, combining resin ducts and terpene
biochemistry. Given the extensive applications of oleoresin products in the chemical,
pharmaceutical and food industries, understanding the molecular basis of terpene
biosynthesis with a view towards improved oleoresin yield and quality is a major goal.
Although oleoresin stimulant pastes have been studied in Pinus elliotti var. elliottii
(slash pine), the underlying gene regulation patterns are unknown. This study evaluated
the expression stability of eleven putative reference genes during oleoresinosis in 16
year-old trees treated with different stimulant pastes on days 0, 5, 8 and 15 after an
event of bark stripping. Single modifications in paste composition of the resin paste
included:
auxin
(1-naphthaleneacetic
acid),
potassium
sulfate
or
2-
chloroethylphosphonic acid (CEPA), along with a control of plain wounding without
paste application. RNA was extracted from exposed cambium near the wounds. Gene
expression was evaluated by geNorm, NormFinder and BestKeeper softwares. Histone 3
and Ubiquitin were identified as the top reference genes and results were consistent with
all algorithms. To validate these results, expression analysis of a putative P. elliotti
orthologue of the P. contorta TPS [(−)-β-pinene synthase] gene was carried out. Upregulation of PcTPS-(−)βpin1 transcripts was noticed after 15 days in trees treated with
CEPA. To our knowledge, this is the first report identifying reference genes for
expression studies during commercial field oleoresinosis in pine, representing a key step
toward unveiling the quantitative transcriptome behind this important non wood forest
product. Financial support: CNPq, CAPES and FAPERGS.