Metabolome profiling of floral scent production in Petunia axillaris
Graphical abstract
Metabolic pathway of floral scent compounds in Petunia axillaris. Compounds whose concentrations showed nocturnal changes are in pink boxes.
Introduction
Due to several excellent properties including ease of growth, short life cycle, relatively compact plant size, and variation in shape and colouration, Petunia plants are often used for physiological studies (Gerats and Vandenbussche, 2005). Strong floral scent is a prominent feature of some Petunia plants, and thus they have become model plants for scent studies (Schuurink et al., 2006). Petunia axillaris, which is widely distributed in temperate South America (Ando et al., 2001), emits scent compounds predominantly at night (Verdonk et al., 2003, Oyama-Okubo et al., 2005), and the mechanisms of regulating floral scent biosynthesis using this property of P. axillaris is a current emphasis. The floral scent compounds of P. axillaris are all benzenoids, and methyl benzoate and iso-eugenol are major components. The nocturnal emission of these floral scent compounds is synchronized with increases in their concentrations in the corolla (Oyama-Okubo et al., 2005). Variations in scent emission strength among the subspecies of P. axillaris are also correlated with corolla concentrations (Kondo et al., 2006). Therefore, based on these previous studies, it is concluded that under constant temperature conditions, the levels of floral scent emission are primarily determined by corolla benzenoid concentrations.
By in vivo stable isotope labeling and computer-assisted metabolic flux analysis, all aromatic compounds in Petunia floral scents were found to be biosynthesized from a common precursor, phenylalanine (Boatright et al., 2004), derived from shikimate and chorismate pathway. Fig. 1 depicts the overall pathway to phenylalanine, including the glucose metabolic pathway which branches after glucose-6-phosphate (G6P) into the glycolysis pathway and the pentose phosphate pathway; these later converge at 3-deoxy-arabinoheptulosonic acid-7-phosphate (DAHP) and lead to phenylalanine as indicated. mRNAs encoding DAHP synthase, 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, chorismate mutase, and phenylalanine ammonia-lyase show rhythmic expression similar to the emission of volatile benzenoids in another strongly scented Petunia, P. hybrida cv. Mitchell (Verdonk et al., 2005). Genes encoding enzymes that catalyse steps in the SAM cycle, including S-adenosyl-l-methionine and benzoic acid/salicylic acid carboxyl methyltransferases were also reported to show similar rhythmic expression (Negre et al., 2003). However, the expression pattern of ODORANT1, a novel R2R3-type MYB transcription factor that regulates expression of the EPSP synthase gene, did not correspond with the increase in volatile benzenoids (Verdonk et al., 2005). Moreover, the expression of many genes encoding methyltransferases that catalyse the synthesis of volatile benzenoids decreased during the night, a finding that is opposite to the emission pattern of volatile benzenoids (Colquhoun et al., 2010). To date, none of the enzymes involved in the biosynthesis of volatile benzenoids have shown significant day–night activity changes (Kolosova et al., 2001, Colquhoun et al., 2010). These findings indicate that major factors other than the expression of biosynthetic genes operate to regulate biosynthesis of volatile scent benzenoids. Here, the possibility was investigated that the activities of some parts of the biosynthetic pathways could be regulated by different substrate concentrations. Changes in the concentration of metabolites from sugars to volatile scent benzenoids were thus analyzed over time to find nocturnal increases in levels of compounds and to identify metabolic steps regulated by substrate concentration in the P. axillaris corolla.
Among wild type P. axillaris plants, there are lines that emit different amounts of scent (Kondo et al., 2006). Thus, to understand the regulation of volatile scent benzenoid biosynthesis in more detail, levels of metabolites were compared between a strongly scented line and a weakly scented line; it was hypothesized that a metabolic interaction between parts of the biosynthetic pathways could be revealed by this comparison. Here, two different P. axillaris lines were selected for study: a strongly scented line, U1AXI, and a weakly scented line, B1320AXI10. Endogenous levels of floral scent compounds in the strongly scented line have diurnal oscillations with maxima at 0000 (midnight) and minima at 1200 (noon) (Oyama-Okubo et al., 2005). In the weakly scented line, the concentration of scent compounds including dihydroconiferyl acetate, a biosynthetically related analog of a scent benzenoid iso-eugenol, was ca. 2% of the strongly scented line (Kondo et al., 2007). Therefore, it was considered that the inhibited step in the weakly scented line occurred earlier than in the pathway for the biosynthesis of scent benzenoids, and that these two lines were suitable materials for probing the regulatory mechanism.
Recently, metabolite profiling (metabolomics) of extracts from numerous species of organisms have been performed by using one or more analytical instruments, such as gas chromatography–mass spectrometry (GC–MS) (Fiehn et al., 2000), capillary electrophoresis–mass spectrometry (CE–MS) (Soga et al., 2003, Soga et al., 2007), liquid chromatography–mass spectrometry (LC–MS) (Yoshida et al., 2007), Fourier transform ion cyclotron resonance mass spectrometry (Aharoni et al., 2002) and nuclear magnetic resonance spectrometry (Reo, 2002). In this regards, the precursors of scent compounds include neutral carbohydrates, phosphorylated saccharides, organic acids, and amino acids. Among these various types of compounds, phosphorylated saccharides are the most difficult to analyse due to their high polarity and negative charges. This problem was resolved by developing a CE–MS system, and for this study, organic acids and amino acids were analyzed by this method. Neutral carbohydrates and scent compounds were also studied by high-performance liquid chromatography (HPLC) and GC–MS, respectively, depending on their chemical properties.
Section snippets
Concentrations of scent compounds
Scent compounds extracted from corollas of strongly (U1AXI) and weakly scented (B1320AXI10) lines of P. axillaris were analyzed qualitatively by GC–MS. Quantitative analyses were performed by GC–FID. Good sensitivity was obtained (Table S1). In the strongly scented line, seven kinds of scent compounds, benzaldehyde, benzyl alcohol, benzyl benzoate, iso-eugenol, methyl benzoate (MB), phenylacetaldehyde and 2-phenylethanol, were detected in corollas, where higher night-time concentrations were
Discussion
To our knowledge, the only sensual property of all P. axillaris lines that is significantly different between day and night-time is floral scent strength; there are no differences in morphology, nyctinasty or colouration. Therefore, it is presumed that compounds whose concentrations show periodic day–night changes in opened corollas are associated with the biosynthesis of floral scent compounds. Other than floral scent strength, no other significant difference between the flowers of these two
Conclusions
The results herein indicate the involvement of varying substrate concentrations in regulating the biosynthesis of benzenoid compounds to generate the nocturnal rhythm of floral scent in P. axillaris corollas. Concentrations of SAM cycle compounds are also affected by concentrations of substrates as methyl-group acceptors. Putative inhibitory steps to reduce the concentrations of floral scent compounds and their multiple influences in some biosynthetic steps in the weakly scented line were also
Plant materials and growth conditions
Plants of P. axillaris lines (strongly scented line; U1AXI, weakly scented line; U1320AXI10) (Kondo et al., 2007) were vegetatively propagated in a greenhouse 2 months before the experiments. Just before the experiments, the plants were acclimated for at least a week in a growth chamber at a constant temperature of 25 °C under a photosynthetic photon flux density of about 250 μmol m−2 s−1 and a 12/12 h (0600–1800 h light/1800–0600 h dark) photoperiod. Flowers were harvested every 6 h starting at 0600 of
Acknowledgments
We thank Prof. Marchesi of University of the Republic (Uruguay) for offering plant materials, Prof. Adachi of Yamaguchi University for offering DQA and DQH, Dr. Ichimura for analyzing carbohydrates and Mr. Kashimura for assistance with plant growth at NIFS. We also thank Dr. Sugimoto, Mr. Hirayama and Ms. Ohishi of Keio University for software development and technical support. Research funds for CE–MS analysis were provided by the Yamagata prefectural Government and Tsuruoka City.
References (29)
- et al.
Reproductive isolation in a native population of Petunia sensu Jussieu (Solanaceae)
Ann. Bot.
(2001) - et al.
Petunia floral volatile benzenoid/phenylpropanoid genes are regulated in a similar manner
Phytochemistry
(2010) - et al.
A model system for comparative research: Petunia
Trends Plant Sci.
(2005) - et al.
Effect of diurnal sampling on the headspace composition of detached Nicotiana suaveolens flowers
Phytochemistry
(1993) - et al.
Regulation of volatile benzenoid biosynthesis in petunia flowers
Trends Plant Sci.
(2006) - et al.
Analysis of nucleotides by pressure-assisted capillary electrophoresis–mass spectrometry using silanol mask technique
J. Chromatogr. A
(2007) An integrated method for spectrum extraction and compound identification from gas chromatography/mass spectrometry data
J. Am. Soc. Mass Spectrom.
(1999)- et al.
Regulation of floral scent production in petunia revealed by targeted metabolomics
Phytochemistry
(2003) - et al.
Nontargeted metabolome analysis by use of Fourier transform ion cyclotron mass spectrometry
OMICS
(2002) - et al.
MathDAMP:pacage for differential analysis of metabolite profiles
BMC Bioinform.
(2006)
Understanding in vivo benzenoid metabolism in petunia petal tissue
Plant Physiol.
Influence of green leaf herbiwory by Manduca sexta on floral volatile emission by Nicotiana suaveolens
Plant Physiol.
Metabolic profiling for plant functional genomics
Nat. Biotechnol.
Soluble carbohydrates in Delphinium and their influence on sepal abscission in cut flowers
Physiol. Plant.
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These authors contributed equally to this work.
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Present address: Horticultural Experiment Station, Yamagata General Agricultural Research Center, 423 Shima-Minami, Sagae, Yamagata 991-0043, Japan.