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© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for 
Experimental Biology. 
This is an Open Access article distributed under the terms of the Creative Commons Attribution 
Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-
commercial re-use, distribution, and reproduction in any medium, provided the original work is 
properly cited. For commercial re-use, please contact journals.permissions@oup.com 
Distinct metabolic pathways drive monoterpenoid biosynthesis in a natural 
population of Pelargonium graveolens (rose scented geranium) 
 
Matthew E. Bergmana, Angel Chavezb, Albert Ferrerb,c, Michael A. Phillipsa,d* 
 
a Department of Cellular and Systems Biology, University of Toronto, Toronto, Ontario M5S 
3G5, Canada 
b Plant Metabolism and Metabolic Engineering Program, Center for Research in Agricultural 
Genomics, (CRAG) (CSIC-IRTA-UAB-UB), Campus UAB, Bellaterra (Cerdanyola del Vallès), 
Barcelona, Spain 
c Department of Biochemistry and Physiology, Faculty of Pharmacy and Food Sciences, 
University of Barcelona, Barcelona, Spain  
d Department of Biology, University of Toronto – Mississauga, Mississauga, Ontario, L5L 1C6 
Canada 
* To whom correspondence should be addressed (michaelandrew.phillips@utoronto.ca) 
 
Highlight: Isotopic labeling studies indicate rose-scented geraniums biosynthesize volatiles 
through distinct cyclic and acyclic monoterpenoid pathways and use (+)-piperitone as an 
intermediate for p-menthane biosynthesis. 
 
  
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Abstract 
Pelargonium graveolens is a wild predecessor to rose-scented geranium hybrids prized for their 
essential oils used as fragrances and flavorings. However, little is known about their 
biosynthesis. Here we present metabolic evidence that at least two distinct monoterpene 
biosynthetic pathways contribute to their volatile profiles; namely, cyclic p-menthanes such as (-
)-isomenthone and acyclic monoterpene alcohols such as geraniol and (-)-citronellol and their 
derivatives (referred to here as citronelloid monoterpenes). We established their common origin 
via the 2C-methyl-D-erythritol-4-phosphate pathway but found no indication these pathways 
share common intermediates beyond geranyl diphosphate. Untargeted volatile profiling of 22 
seed-grown P. graveolens lines demonstrated distinct chemotypes that preferentially accumulate 
either (-)-isomenthone, geraniol, or (-)-citronellol along with approximately 80 minor volatile 
products. Whole plant 13CO2 isotopic labeling performed under physiological conditions 
permitted us to measure the in vivo rates of monoterpenoid accumulation in these lines and 
quantify differences in metabolic modes between chemotypes. We further determined that p-
menthane monoterpenoids in Pelargonium are likely synthesized from (+)-limonene via (+)-
piperitone rather than (+)-pulegone. Exploitation of this natural population enabled a detailed 
dissection of the relative rates of competing p-menthane and citronelloid pathways in this 
species, providing real time rates of monoterpene accumulation in glandular trichomes. 
 
Keywords 
Essential oils, Geraniaceae, glandular trichomes, monoterpenoid biosynthesis, untargeted 
metabolomics, volatile profiling, isotopic labeling 
 
Abbreviations used: 
C:G  Citronellol:geraniol ratio 
DMADP Dimethylallyl diphosphate 
EO Essential oil 
GC-MS Gas chromatography – mass spectrometry 
GDP  Geranyl diphosphate 
GT Glandular trichomes 
MEP  2C-Methyl-D-erythritol-4-phosphate 
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MVA Mevalonate 
IDP Isopentenyl diphosphate  
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Introduction 
Scented geraniums (Pelargonium sp.) are an aromatic genus of glandular trichome (GT) 
bearing plants in the family Geraniaceae noted for their terpenoid rich essential oil (EO), a 
feature which has made them useful in the flavor, perfume, and fragrance industries. Several 
wild species, including P. capitatum, P. graveolens, and P. radens contributed to modern 
cultivars as a result of centuries of complex hybridization by European breeders using specimens 
collected around the Cape area of South Africa during the 18th and 19th centuries (Demarne and 
Van der Walt 1989; Tucker and Debaggio 2009; Lis-Balchin et al. 2003). These scented 
geraniums are close relatives to the more widely cultivated regal pelargoniums (P. domesticum) 
and zonal geraniums (P. x hortorum) (Loehrlein and Craig 2001; Loehrlein and Craig 2000). The 
term ‘Graveolens cultivar group’ is sometimes used to distinguish so-called rose-scented 
geraniums with clear P. graveolens heritage, but rampant hybridization has resulted in complex 
lineages of unclear ancestry. Commercial hybrids are widely cultivated for the perfumery, 
flavoring and cosmetics industries (Blerot et al. 2016), but EO from rose-scented geraniums has 
also attracted interest for its efficacy as an anti-microbial (Lis-Balchin and Deans 1997; Lis-
Balchin et al. 1998; Nadjib Boukhatem et al. 2013) and acaricidal food preservative (Jeon et al. 
2009), as a fumigant (Baldin et al. 2015; Seo et al. 2009) and for its hypoglycemic and anti-
oxidant properties (Chen and Viljoen 2010; Boukhris et al. 2015; Boukhris et al. 2012).  
 Pelargonium EO consists primarily of oxygenated C10 monoterpenoid volatiles with 
variable amounts of C15 sesquiterpenes. While the monoterpenoid fraction of Pelargonium EO 
varies according to the cultivar and season (Verma et al. 2013), it consists primarily of acyclic 
monoterpene alcohols geraniol and (-)-citronellol (and their aldehyde, acid, and ester derivatives, 
referred to collectively here as ‘citronelloid monoterpenes’), oxygenated p-menthanes such as 
(+)-menthone and (-)-isomenthone, and lesser amounts of olefinic hydrocarbons such as 
limonene and -phellandrene (Babu and Kaul 2005; Blerot et al. 2016; Karami et al. 2015) (Fig. 
1). These volatile compounds are thought to accumulate in type 1 and 2 capitate glandular 
trichomes on the leaf surface (Boukhris et al. 2013a). Phytochemical analysis has suggested the 
total volatile complement of Pelargonium consists of more than 100 features, the bulk of which 
are terpenoids (Jain et al. 2001; Kulkarni et al. 1998; Shellie and Marriott 2003; Wang et al. 
2014).  
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Plant breeders have focused on cultivars rich in geraniol and (-)-citronellol as they impart 
a pleasing rosy fragrance (Verma et al. 2013) while cultivars with a higher (-)-isomenthone 
content (Kulkarni et al. 1998) present minty aromas with lower market value. Wild type P. 
graveolens often features abundant (-)-isomenthone (Lalli et al. 2006), and breeding efforts have 
resulted in a shift towards strains higher in citronelloids and lower in p-menthane monoterpenes. 
A low citronellol:geraniol (C:G) ratio is an important determinant of geranium EO quality as 
well as an indicator of its geographical origin. This ratio varies with ambient temperature in field 
grown cultivars, but the relationship is not straightforward. Warmer months favoured citronellol 
and its esters over geraniol in the Graveolens group Bourbon cultivar (Rajeswara Rao et al. 
1996). However, a high C:G ratio also correlates with low night time temperatures in the 
Graveolens G1 clone (Doimo et al. 1999). Interestingly, citronelloid dominant cultivars readily 
revert to a wild-type, isomenthone-rich phenotype as a result of somatic mutagenesis (Kulkarni 
et al. 1998; Saxena et al. 2004; Gupta et al. 2001). Thus, the C:G ratio in citronelloid rich 
cultivated varieties is under environmental control while the prevailing structural group (i.e. 
citronelloids or p-menthanes) is evidently controlled at the genetic level.  
Essentially all monoterpenes are derived from geranyl diphosphate (GDP), itself the 
product of two C5 units derived from the 2C-methyl-D-erythritol-4-phosphate (MEP) pathway in 
the plastid (Banerjee and Sharkey 2014; Phillips et al. 2008; Frank and Groll 2017). The MEP 
pathway synthesizes isopentenyl and dimethylallyl diphosphate (IDP and DMADP) from 
glyceraldehyde-3-phosphate and pyruvate in a 7-step process which in turn provides the 
precursors for the synthesis of primary terpenoid metabolites including chlorophyll and 
prenylquinones (Hoeffler et al. 2002; Kim et al. 2013; Liu and Lu 2016), phytohormones 
(Hedden and Thomas 2012; Sakakibara 2006; Nambara and Marion-Poll 2005), and carotenoids 
(Ruiz-Sola and Rodriguez-Concepcion 2012) in addition to a diverse suite of secondary 
terpenoid metabolites (Gershenzon and Dudareva 2007). IDP and DMADP can also be formed 
through the cytosolic mevalonate (MVA) pathway, which utilizes acetyl-CoA as a carbon source 
and supplies the synthesis of mainly sesquiterpenes (C15) and triterpenes (C30) (Bach et al. 1999). 
In some plant lineages, terpenoid secondary metabolism is associated with GTs. For example, the 
MEP pathway supplies monoterpene biosynthesis in GTs in members of the Geraniaceae (Blerot 
et al. 2016), Cannabaceae (Champagne and Boutry 2017; Booth et al. 2017), Solanaceae (Balcke 
et al. 2017), and Lamiaceae (McCaskill et al. 1992; Hallahan 2000).  
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The peppermint (Mentha x piperita) (-)-menthol pathway is currently the best 
characterized system for understanding monoterpene biosynthesis in GTs, an elaborate multi-
step pathway spanning at least 3 subcellular compartments (reviewed in (Croteau et al. 2005; 
Lange 2015)). Briefly, GDP is cyclized into (-)-limonene in the plastid by (-)-limonene synthase 
followed by its oxidation to (-)-trans-isopiperitenol in the endoplasmic reticulum by (-)-
limonene-3-hydroxylase. This intermediate reaches the mitochondria where (-)-trans-
isopiperitenol dehydrogenase oxidizes it into (-)-isopiperitenone. In the cytosol, reduction of the 
double bond by (-)-isopiperitonene reductase yields (+)-cis-isopulegone which is then isomerized 
into (+)-pulegone, the penultimate precursor before (-)-menthol formation. (+)-Pulegone 
reduction by NADPH-dependent (+)-pulegone reductase yields (-)-menthone and less amounts of 
(+)-isomenthone. Reduction of these cycloketones to (-)-menthol and (+)-isomenthol, 
respectively, occurs in the cytosol, although (-)-menthol is the principal end product.  
By contrast, little is known about the biosynthesis of the corresponding compounds in 
Pelargonium. Peppermint and Pelargonium share two p-menthane cycloketones in common but 
the stereochemistry is inverted (Pelargonium accumulates (+)-menthone and (-)-isomenthone) 
(Ravid et al. 1994). Blerot et al. recently described four terpene synthases from P. graveolens, 
including a geraniol synthase and the sesquiterpene synthase responsible for 10-epi--eudesmol 
(Blerot et al. 2018). The precursor to (-)-isomenthone remains unknown in P. graveolens. It is 
likewise unclear whether citronelloid and p-menthane monoterpenes share common biosynthetic 
intermediates downstream of GDP in this species or whether they constitute independent 
pathways competing for a common pool of GDP. Here we have applied isotopic labeling studies 
and untargeted metabolomic analysis to a natural population of P. graveolens consisting of well-
defined chemotypes which preferentially accumulate geraniol, (-)-citronellol, or (-)-isomenthone. 
With this approach, we observed the functional independence of p-menthane and citronelloid 
monoterpene biosynthesis in closely related P. graveolens genetic backgrounds and inferred 
previously unknown features of this biosynthetic network. For instance, unlike the related (-)-
menthol pathway in peppermint, we present evidence that P. graveolens p-menthane 
biosynthesis utilizes (+)-piperitone as a biosynthetic intermediate in place of (+)-pulegone and 
uses (+)-limonene as a precursor. 
  
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Results and discussion 
Volatile profiling in a P. graveolens natural population suggests mutually exclusive p-menthane 
and citronelloid specialists 
We compared the volatile profiles of 22 seed grown individuals from a wild population of 
P. graveolens to identify possible chemotypes. Gas chromatography – mass spectrometry (GC-
MS) analysis of P. graveolens volatiles was carried out on trichome-rich, expanding leaf tissue. 
Untargeted profiling identified a total of 322 volatile features among all lines. From this group, 
89 were reproducibly detected within the individual plant lines. Principal component analysis of 
the 22 lines was carried out using these 89 volatile compounds as input variables (Fig. 2). 
Approximately 77% of the variation was explained by the first component, while the second 
accounted for another 20%. Lines positively correlated with the first component were highly 
enriched in the p-menthane monoterpene (-)-isomenthone (15 lines), while lines correlated 
negatively with the first component demonstrated enrichment with citronelloid monoterpenes 
such as geraniol (2 lines), and (-)-citronellol (5 lines). Thus, the single most defining factor in the 
volatile profile of a given line was whether it was dominated by cyclic p-menthane type or 
acyclic citronelloid type monoterpenes. P. graveolens lines enriched in the citronelloid 
monoterpenes geraniol and (-)-citronellol (and, to a lesser extent, (-)-citronellal), varied 
principally along the second component (i.e., they could be distinguished by their C:G ratios). 
No plant line was observed which contained equal proportions of p-menthane and citronelloid 
monoterpenes. These four volatile compounds account for most of the differences among the 
volatile profiles of these lines (Fig. 2). The single citronellic acid enriched line, Pg28, differed 
from the other four lines comprising the (-)-citronellol clade in that it lacked the aldehyde form 
citronellal but instead presented significant level of the corresponding acid. A typical 
chromatogram of an (-)-isomenthone rich line showed that upwards of 72.2% (± 3.7%) of the 
total integrated peak area was due to this compound (Fig. 3), corresponding to an absolute 
concentration of 11.11 ± 0.91 mg∙g-1 F.W., followed by trace levels of other volatiles, most 
prominently β-myrcene and trans-β-ocimene, which made up 3.7% (± 1.6%) and 2.6% (± 0.8%) 
of the total integrated peak area, respectively. (-)-Citronellol rich lines displayed greater 
diversity, with (-)-citronellol comprising 35.1% (± 12.0%) of the total peak area (corresponding 
to 5.02 ± 0.37 mg∙g-1 F.W.), (-)-isomenthone 21.0% (± 5.7%) and the remainder contributed by 
multiple minor components, of which (-)-citronellal and geranial contributed the largest amount 
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by peak area at 16.1% (± 7.2%) and 1.7% (± 1.2%), respectively (Fig. 3). Finally, the profiles of 
members of the geraniol rich group consisted of approximately 50.5% geraniol (± 12.0%) (4.76 
± 0.96 mg∙g-1 F.W.), followed by 15.4% (± 10.4%) --isomenthone and 11.4% (± 3.3%) (-)-
citronellol as its major components (Fig. 3). For comparative purposes, the Graveolens cultivar, 
derived largely from P. graveolens as well as P. radens and P. capitatum, was similarly analyzed 
and found, unlike the natural chemotypes, to contain more balanced levels of p-menthane and 
citronelloid monoterpenes. Comprehensive lists of volatile compounds in P. graveolens and its 
cultivars have been published previously (Jain et al. 2001; Wang et al. 2014; Shellie and Marriott 
2003; Kulkarni et al. 1998) and reviewed (Blerot et al. 2016). While our results are in general 
agreement with these volatile profiles, we have chosen to focus on the ~6-8 volatile compounds 
which define differences between chemotypes and typically make up >95% of the total ion 
chromatogram by peak area. 
When hierarchical clustering was applied to these same data, the separation of (-)-
isomenthone, geraniol, and (-)-citronellol rich chemotypes was evident with 15 of the 22 lines 
falling into the (-)-isomenthone rich category and 5 into the (-)-citronellol group (Fig. 4A). In 
this case, the Pg28 line clustered as a singleton within the (-)-citronellol group, while all other 
clusters consisted of at least two independent lines. The remaining two lines contained geraniol 
as their principal volatile compound. These groupings matched our principal component analysis 
data that showed only 3-4 volatile compounds were responsible for ~97% of the variation in the 
data set. The (-)-isomenthone cluster could be subdivided further, mainly due to small 
differences in low abundance sesquiterpenes and olefinic monoterpenes. However, the cluster 
height of these relationships in the dendrogram did not justify further division into smaller 
clusters. The entire analysis was repeated approximately six months later to determine if seasonal 
or other environmental factors played a role in the observed volatile profiles. However, the 
relative enrichments and overall compositions were essentially identical across all seed grown 
lines surveyed. From these observations, we conclude that P. graveolens natural populations 
consist of phenotypes characterized by a consistent volatile chemical profile that is stable across 
phenological variation and generation. Furthermore, these chemotypes are dominated either by 
the p-menthane monoterpene (-)-isomenthone or by one of the citronelloid monoterpene alcohols 
(geraniol or (-)-citronellol). An inspection of the gross characteristics of these chemotypes did 
not reveal obvious differences in leaf morphology (Supplemental Fig.1). Breeding efforts which 
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have produced commercially important varieties such as Algerian, Bourbon, and Kelkar have 
focused on lines enriched in citronelloid monoterpenes, and genotypic variation in the relative 
amounts of their acyclic monoterpene alcohols is well established (Rao 2009). However, our 
results demonstrate that the parental lines themselves which gave rise to modern rose-scented 
geranium cultivars featured distinct chemotypic groups which can be distinguished, firstly, by 
the presence or absence of (-)-isomenthone as the dominant volatile component and, secondly, 
for citronelloid rich lines, by their C:G ratios. The absence of any line with comparable levels of 
p-menthane and citronelloid monoterpenes suggests that in wild type plants, one pathway tends 
to predominate. We only observed comparable levels of p-menthane and citronelloid 
monoterpenes in the Graveolens cultivar hybrid line. 
 We next examined correlations between principle volatile components and the 22 wild-
type lines to infer details about their biosynthetic relationships. The heatmap in Fig. 4B indicates 
that in addition to the expected high abundance of (-)-isomenthone, members of the (-)-
isomenthone clade also displayed higher levels of other p-menthane monoterpenes including 
limonene, (+)-menthone and (+)-piperitone as well as a near complete absence of (-)-citronellol 
and geraniol. The geraniol rich clade demonstrated a corresponding reduction in the cyclic p-
menthanes limonene, (+)-menthone, and (-)-isomenthone as well as a high degree of enrichment 
of geranyl esters (principally formate, acetate, and tiglate) (Fig. 4B). Finally, the corresponding 
citronellyl esters were highly enriched in the (-)-citronellol clade, analogous to those observed in 
the geraniol group, and this clade was also characterized by an absence of p-menthane type 
monoterpenes and variable levels of (-)-citronellal.  
A correlation matrix generated from these data (Fig. 4C) demonstrated the co-occurrence 
of several groups of monoterpene volatiles according to their structural class, suggestive of 
membership in a common biosynthetic sequence. For instance, the p-menthane monoterpenes 
limonene, menthone, isomenthone, and piperitone (Fig.1) all showed a strong correlation (>0.5 
Pearson’s correlation coefficient). Similarly, citronelloid monoterpenes including geraniol, (-)-
citronellol, geranial, (-)-citronellal, citronellic acid, and citronellyl and geranyl esters showed a 
strong correlation to other acyclic monoterpenes (Fig. 4C). At the same time, p-menthane rich 
lines showed a strong negative correlation (< -0.5 Pearson’s correlation coefficient) with 
citronelloids and vice versa. The positive correlation between limonene and (-)-isomenthone 
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suggest the former may serve as precursor for p-menthane biosynthesis in Pelargonium, in 
keeping with a similar role for limonene as a precursor for p-menthane biosynthesis in the 
peppermint (-)-menthol pathway (Lange 2015). The structural similarity of piperitone to 
isomenthone (Fig. 1), along with the strong co-occurrence observed in this analysis, are 
suggestive of a precursor-product relationship. At the same time, the strong negative correlation 
between citronelloids and p-menthane in these wild-type lines indicates these two metabolic 
specializations may be largely mutually exclusive in natural populations.  
Inhibitors treatments confirm the MEP pathway supplies monoterpene biosynthesis in 
Pelargonium 
To determine the principal precursor pathway responsible for providing isoprene 
equivalents for monoterpene biosynthesis in P. graveolens, Graveolens cultivar plants were 
sprayed once per day with either an inhibitor of the chloroplast localized MEP pathway 
(clomazone, which is converted in planta to ketoclomazone, a potent DXS inhibitor (Matsue et 
al. 2006)), mevinolin, which inhibits the HMG-CoA reductase step of the MVA pathway 
(Alberts et al. 1980), or a control solution. The rate of synthesis of plastid-derived terpenes, as 
judged by 13C incorporation, should be reduced following clomazone treatment, whereas 
cytosolic terpenes derived from the MVA pathway should be affected by mevinolin treatment. 
After 72 hours of inhibitor or control treatment, plants were subjected to 13CO2 isotopic labeling 
assays as described above and harvested at a single time point of 6 hours. We observed decreases 
in the rate of incorporation of 13C into monoterpenoid end product pools when plants were 
sprayed with clomazone (Fig. 5), but not when treated with mevinolin or a control solution. From 
these observations, we conclude that the MEP pathway supplies precursors for monoterpene 
biosynthesis in P. graveolens. 
 
Isotopic labeling studies in intact plants reveal physiological rates of monoterpenoid volatile 
accumulation in P. graveolens 
We carried out 13CO2 labeling assays on intact plants to measure the rate of 
13C 
incorporation into various monoterpenoid volatile components and to gain insight into their 
biosynthetic relationships. Identical 4-6 month old rooted cuttings of the ‘Graveolens’ cultivar 
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were incubated in air containing 13CO2 at 400 p.p.m. for 1 – 9 h following a 1 h adaptation phase 
in standard air in an environmentally controlled dynamic flow cuvette. Analysis of the resulting 
labeled organic extracts by GC-MS showed detectable levels of 13C in major oil components of 
the Graveolens cultivar such as geraniol, (-)-isomenthone, limonene, and (+)-piperitone in as 
little as 3 h (Fig. 6). Volatile components showing detectable label incorporation on this time 
scale were selected for closer analysis. Using an optimized GC-MS SIM method, we calculated 
the fractional labeling of each feature at each labeling time point between 0 and 9 h using the 
relative m/z intensities of the molecular ion cluster representing the unlabeled (M+0) and various 
isotopologs of each C10 volatile compound (M+1 through M+8). These data were then compared 
to the absolute concentration as judged by comparison to external calibration curves. The 
combined data allowed us to express the moles of 13C equivalents detected in each metabolite 
pool over time.  
This whole plant isotopic labeling approach permitted us to measure the rate of 
monoterpenoid accumulation in glandular trichomes under physiological conditions in intact 
plants. The volatile profile of the Graveolens cultivar consists of significant amounts of geraniol, 
(-)-citronellol, and (-)-isomenthone (Fig. 3) and thus provides a useful starting point to 
collectively evaluate the relative rates of the three major end products in a single plant line. We 
observed the highest rate of label incorporation into geraniol and (-)-isomenthone in this line, 
which became labeled at a rate of 278 and 250 pmol 13C∙mg-1 FW∙h-1, respectively (Fig. 6). This 
rapid rate of labeling would be consistent with a direct formation of geraniol from GDP. 
However, a comparable rate of label incorporation into (-)-isomenthone, which is expected to 
undergo a longer series of transformations from GDP was surprising. (-)-Citronellol and (+)-
piperitone both showed significantly lower rates of incorporation in this hybrid line at 56 and 65 
pmol 13C∙mg-1 FW∙h-1, respectively, but the lowest incorporation rate was observed for limonene 
at only 29 pmol 13C∙mg-1 FW∙h-1.  
We then applied this whole plant time course labeling approach to plants representing the 
three major chemotypes to observe the synthesis of p-menthane and citronelloid monoterpenes in 
isolation. To do this, we relied on a -cyclodextrin capillary GC column which is capable of 
resolving simple enantiomeric pairs. It should be noted that due to improvements in growth and 
labeling conditions used in wild-type labeling assays, these results cannot be directly compared 
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to those of the Graveolens cultivar. Differences in flux towards p-menthane and citronelloid 
monoterpenes were readily observed among the three main chemotypes (table 1). For instance, in 
p-menthane ((-)-isomenthone) enriched chemotypes, 13C label appeared in the (-)-isomenthone 
pool beginning at 3 h and increased at a rate of 1,719 pmol 13C∙mg-1 FW∙h-1 (Supplemental Fig. 
S2), whereas the flux of 13C into (-)-isomenthone in the citronellol and geraniol rich chemotype 
was only 218 and 70 pmol 13C mg-1 FW∙h-1, respectively. In contrast, label appeared above 
background levels in only 2 h in the geraniol pool of the geraniol-rich chemotype, increasing at a 
rate of 381 pmol 13C mg-1 FW∙h-1 (and undetectable in (-)-citronellol and (-)-isomenthone-rich 
lines).  
Quantification of other p-menthane intermediates in the (-)-isomenthone-rich strain was 
also feasible with this approach. Increases in label were detected in (+)- and (-)-limonene at rates 
of 72 pmol 13C∙mg-1 FW∙h-1 and 13 pmol 13C∙mg-1 FW∙h-1, respectively, while (+)-piperitone 
labeling was observed at a rate of 849 pmol 13C∙mg-1 FW∙h-1. Geraniol was present only at trace 
levels in this chemotype, and 13C label in this metabolite pool was indistinguishable from 
background levels. These observations, together with correlation data (Fig. 4), lead us to 
conclude that p-menthane and citronelloid biosynthesis operate independently, rely on the same 
GDP precursor pool, and do not appear to share any downstream intermediates. 
These direct metabolic data impact our understanding of how essential oil biosynthesis 
takes place in P. graveolens, although the interpretation is not straightforward. If limonene 
serves as precursor to (-)-isomenthone via (+)-piperitone, our initial expectation was that 
upstream intermediates should show an incorporation rate at least as high as downstream 
intermediates in order to sustain the needed supply. That this was not the case may be explained 
by several possibilities. Firstly, the precursor for (-)-isomenthone might be something other than 
limonene, despite strong correlation and heat map data that suggest their membership in a 
common biosynthetic sequence. However, these results may also be explained by significant 
back reactions for reversible steps, significant departure from true steady state conditions, or 
long range transport of certain intermediates as glycosides (Wüst et al. 1999) and their 
subsequent deconjugation, could also explain the observed labeling patterns. Moreover, multiple 
transport processes, cell types, and central metabolic pathways are involved in the biosynthesis 
of these labeled natural products which together could introduce unpredictable effects on label 
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distribution. These include the fixation of 13CO2 in photosynthetic cells via the Calvin Benson 
cycle, conversion to sucrose or other sugars, transport to the leucoplasts of glandular trichomes 
(Turner et al. 1999), partial degradation via glycolysis to form pyruvate and glyceraldehyde 3-
phosphate, conversion to IDP and DMADP through the MEP pathway, assembly of GDP, and 
then as of yet uncharacterized steps culminating in the formation of labeled monoterpenoid end 
products. Unequal loss of some intermediates through volatilization may further skew these 
results. The transport to the subcuticular storage space of captitate glandular trichomes (Tissier et 
al. 2017) represents an additional step which may complicate monoterpenoid labeling, although 
our analytical system does not distinguish between intercellular labeled monoterpenes awaiting 
transport and those in the storage cavity. Finally, glycosylation and transport of some 
intermediates may partly contribute to these observations. While little is known regarding 
terpene glycoside formation in P. graveolens compared to the glycosylation of flavonoids 
(Boukhris et al. 2013b), conjugation of terpene alcohols to sugar residues is well known in other 
plant systems (Houshyani et al. 2013; Andrade et al. 2018). Future studies taking conjugation 
into account and diversion of photosynthate into sequestered glycoside pools will be necessary to 
build comprehensive models for the biosynthesis of these metabolites. 
Despite the inconsistencies in precursor-product labeling patterns in limited cases, these 
results overall provide useful insights into the kinetics of multiple processes linking primary and 
secondary metabolism. For example, we have shown that 13C label, starting from 13CO2, is 
detectable in functionalized monoterpenoid end products such as (-)-isomenthone in as little as 
~2 h in illuminated plants, and less for geraniol. Previous reports of 13CO2 whole plant volatile 
labeling have focused on emitted monoterpenes in herbivory stressed cotton (Pare and 
Tumlinson 1997) or oak (Loreto et al. 1996), or isoprene emissions in oak and poplar (Delwiche 
and Sharkey 1993; Karl et al. 2002; Ghirardo et al. 2014; Loreto et al. 2004). However, the 
present report may constitute the first direct kinetic assessment of carbon flux into stored 
monoterpenoids in the GTs of intact plants. 
 
(+)-Limonene is the precursor to the p-menthane monoterpene biosynthesis Pelargonium 
The asymmetrical incorporation patterns of (+) and (-)-limonene in the (-)-isomenthone 
chemotype (table 1) led us to further investigate the precursor to (-)-isomenthone. Unlike 
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peppermint, which accumulates almost exclusively (-)-limonene as a precursor to (-)-menthol 
biosynthesis or lemon grass (Cymbopogon citratus) which accumulates mostly the (+) 
enantiomer, P. graveolens EO from the two citronelloid chemotypes features a nearly racemic 
mixture of the (+) and (-) forms of limonene (Fig. 7A). In the case of the (-)-isomenthone 
chemotype, this ratio is skewed towards (+)-limonene (63%). Most plant species typically favor 
one enantiomer over the other in the case of chiral natural products. However, exceptions to the 
rule exist. Pine produces significant quantities of (+)- and (-)--pinene (Phillips et al. 1999) for 
example. In this case, there is a dedicated terpene synthase responsible for each enantiomer 
(Phillips et al. 2003) rather than a single terpene synthase capable of producing both. Since 
limonene is the most abundant monoterpene hydrocarbon in P. graveolens and because it serves 
as precursor to the p-menthane monoterpenes in peppermint, we examined carbon flux into (+)- 
and (-)-limonene to determine whether the observed differences in pool size of these two 
enantiomers among chemotypes were paralleled by chemotypic differences in carbon flux. 
Percent atom labeling analysis of the two limonene enantiomers in the (-)-isomenthone rich lines 
demonstrated that 81% of the observed limonene flux goes through the (+) enantiomer (Fig. 7C). 
The citronelloid-rich lines demonstrated far lower levels of limonene overall, and the isotopic 
enrichment did not favor (+)-limonene to the extent seen in (-)-isomenthone rich chemotypes 
(Fig. 7B). The absolute rate of 13C incorporation into (+)-limonene (72 pmol 13C∙mg-1 FW∙h-1) in 
the (-)-isomenthone rich line was >20 times the rate of flux into (+)-limonene in geraniol rich 
lines, which partly reflects the smaller pool size of limonene in this line, and no flux was 
detected into (+)-limonene in the (-)-citronellol chemotype whatsoever (table 1). Therefore, 
given the correlation data above and the stereospecific incorporation of 13C into (+)-limonene, a 
pattern specific to the (-)-isomenthone line, (+)-limonene is currently the best candidate for the 
precursor to (-)-isomenthone biosynthesis in P. graveolens. Further investigation via biochemical 
characterization of recombinant proteins is needed to confirm this preliminary conclusion. 
 
Pulegone is not an intermediate of Pelargonium p-menthane biosynthesis 
We investigated whether pulegone is an intermediate in p-menthane biosynthesis in Pelargonium 
as it is in peppermint. We were unable to detect pulegone by GCMS analysis in scan mode. 
Using a dedicated SIM method for the characteristic ions of pulegone (m/z 152, 81, and 67) and 
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SPME adsorption to minimize background, we observed a barely detectable peak at the expected 
retention time of 16.28 min in Pelargonium (Supplemental Fig. S3), but this was below the limit 
of detection of our analysis. Furthermore, its identity could not be confirmed as pulegone as the 
apices of these mass traces did not align. In contrast, (+)-pulegone was readily detectable in 
peppermint extracts (Supplemental Fig. S3). When we compared this peak in extracts made from 
similar amounts of fresh tissue from each species, the unknown peak in Pelargonium was present 
at nearly 400 fold lower concentration (<0.3% by peak area) than the confirmed (+)-pulegone in 
peppermint, making it unlikely that pulegone, if indeed present in Pelargonium, plays the same 
role in Pelargonium that it does in peppermint. Moreover, when we made a similar comparison 
of the peak corresponding to (+)-piperitone in Pelargonium and peppermint, the situation was 
reversed: Pelargonium showed 60 fold more (+)-piperitone than peppermint per mg FW. 
Furthermore, the (+)-pipertitone pool in Pelargonium showed rapid labeling kinetics, confirming 
it is a rapidly turned over intermediate, while isotope analysis of the putative (+)-pulegone in 
Pelargonium was not possible due to extremely low abundance. Given these observations, we 
conclude that (+)-pulegone, if present at all in Pelargonium, is present at such trace levels that it 
is highly unlikely it participates in the biosynthesis of p-menthane monoterpenes in this species. 
Furthermore, the rapid labeling kinetics of (+)-piperitone and its significantly higher steady state 
concentrations led us to conclude that in Pelargonium, unlike in peppermint, (+)-piperitone 
serves as an upstream intermediate to the formation of (-)-isomenthone, while (+)-pulegone does 
not. 
 
Hierarchical clustering of volatiles in Pelargonium cultivars highlights the division between p-
menthane and citronelloid monoterpenoid biosynthesis  
We analyzed the complete volatile profiles of several well-known Pelargonium hybrids 
and species along with the representative chemotypes described above to gain further insight into 
the organization of the two principal monoterpene biosynthetic networks that characterize this 
genus. In contrast to Fig. 4, this comparison involves more distantly related species. Similar 
comparisons have been carried out for chemotaxonomic purposes (Lalli et al. 2006) although 
molecular phylogeny of the chloroplast, mitochondrial, and nuclear genomes may be more 
accurate (Bakker et al. 2004; Bakker et al. 2000). Hierarchical clustering of 132 distinct volatile 
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compounds from 17 Pelargonium specimens representing wild species, cultivars, or chemotypes 
of P. graveolens demonstrated clustering that was only weakly consistent with their generally 
accepted taxonomic relationships (Fig. 8A and 8B). For instance, the citronellol chemotype 
grouped closely with P. radens due to high levels of (-)-citronellol in both, consistent with 
Bakker et al, but the isomenthone chemotype of P. graveolens grouped closely with P. 
tomentosum due to an abundance of p-menthanes in each, despite molecular approaches placing 
these two species much further apart than P. graveolens and P. radens.  
 Despite the inconformity between chemotaxonomic approaches and molecular 
phylogeny, we nonetheless found that inclusion of a wider variety of Pelargonium species and 
cultivars illustrated other likely biosynthetic intermediates associated with each of the two 
monoterpenoid pathways. For instance, correlation analysis of the 14 most significant features 
suggest piperitol, piperitone, limonene, menthone, and isomenthone, which all bear a p-menthane 
skeleton, share a common biosynthetic pathway (Fig. 8C). On the other hand, the acyclic 
citronelloid monoterpenes (geraniol, neral, geraniol, (-)-citronellol, and the formate esters of 
geraniol and (-)-citronellol) seen in other lines are notably depleted when p-menthane skeletal 
types are abundant. Conversely, citronelloid monoterpenes co-occur strongly and are similarly 
deficient in p-menthane types. This is consistent with the overall division of these two pathways 
and further supports the notion that one or the other tends to predominate in a given species or 
cultivar while examples of comparable proportions of p-menthanes and citronelloids, as seen in 
the Graveolens cultivar, are the exception. 
The structure of the monoterpenoid biosynthetic network in P. graveolens can be partly inferred 
from isotopic labeling studies 
 From these observations, we conclude that monoterpenoid biosynthesis in P. graveolens, 
and likely other members of this genus, can be characterized by variable contributions of two 
pathways which yield either cyclic p-menthane type monoterpenes such as (-)-isomenthone and 
(+)-menthone or acyclic monoterpene alcohols and their derivatives, described collectively here 
as citronelloid monoterpenes. The latter command a higher value in fragrance and flavoring 
industries and are generally more pleasing to the human olfactory palette. Breeders have 
traditionally selected for Pelargonium hybrids enriched in geraniol, citronellol, nerol, and 
depleted in isomenthone. We initially considered whether these cyclic and acyclic monoterpene 
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groups shared common intermediates, for instance through the cyclization of a non-activated 
acyclic precursor. Neral and (-)-citronellal (Fig. 1) possess the correct electronic and geometric 
configurations for direct cyclization into (+)-piperitone and (-)-isomenthone, respectively, via a 
hydride shift. While unusual, a similar reaction has been reported for a promiscuous squalene-
hopene cyclase from the Gram-negative bacterium Zymomonas mobilis capable of converting 
citronellal into isopulegol in bioreactors (Siedenburg et al. 2012). However, our heat map and 
correlation data (Fig.s 4B and 4C) clearly show that the p-menthane and citronelloid skeletal 
types are largely mutually exclusive in wild type lines, a pattern which holds in cultivated 
hybrids as well (Fig. 8), making a direct connection between these two pathways highly unlikely. 
Moreover, the strong correlation we observed between flux into (+)-limonene and flux into (-)-
isomenthone (Fig. 7), an effect absent in (-)-isomenthone deficient lines, supports the functional 
independence of these two pathways with (+)-limonene currently the best candidate for precursor 
to p-menthane monoterpenes in this species. 
We further conclude that in the glandular trichomes of P. graveolens favor p-menthane 
biosynthesis leading to (-)-isomenthone production over citronelloid production in their natural 
state, an effect likely rooted in the metabolic control of these two pathways. This conclusion is 
based on the significant levels of measured flux through the p-menthane pathway even in 
geraniol and (-)-citronellol specializing lines (table 1, Supplemental Fig. S2). In contrast, flux 
towards the citronelloid types in (-)-isomenthone chemotypes was virtually undetectable. The 
overall magnitude of flux also grossly favored (-)-isomenthone production when the calculated 
rates of labeling were compared between the main end products in each of the three chemotypes 
(1,719 pmol 13C mg-1 FW∙h-1 (-)-isomenthone in the (-)-isomenthone chemotype versus 218 and 
70 pmol 13C mg-1 FW∙h-1 (-)-citronellol and geraniol in their respective chemotypes). Such a 
dominant role of the p-menthane pathway in determining product profiles would also be 
consistent with instances of spontaneous reversion of geraniol rich cultivars to isomenthone rich 
lines (Kulkarni et al. 1998; Saxena et al. 2004; Gupta et al. 2001). The identification of protein 
targets to overcome this natural tendency towards (-)-isomenthone production would be of value 
to breeders aiming to maximize geraniol and (-)-citronellol production, as well as their highly 
valued esters. Similar approaches to improving EO quality in peppermint relied on the 
identification of the menthofuran synthase transcript, whose protein product diverts flux towards 
the undesirable product (+)-menthofuran (Bertea et al. 2001). Suppression of this transcript 
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enabled a reduction of (+)-menthofuran content and improvement of oil quality (Mahmoud and 
Croteau 2003). 
The use of volatile profiling to define chemotypes has also been applied to oregano 
(Thompson et al. 2003), and these chemotypes were central to the elucidation of the biosynthetic 
pathway of phenolic monoterpenes in this species (Crocoll et al. 2010). The present work 
identifies future targets for cloning and biochemical characterization and provides a context for 
understanding the significance of essential oil genes identified to date. Blerot et al. (2019) 
recently characterized four terpene synthases from P. x hybridum, including the enzyme 
responsible for the formation of geraniol from GDP, but no enzyme from this genus responsible 
for its reduction to (-)-citronellol has yet been identified. Indeed, Wuest et al suggested that P. 
graveolens uses citronellyl diphosphate as a precursor to rose oxide (Wüst et al. 1999), a high 
value monoterpene volatile structurally similar to citronellol, and this substrate could potentially 
serve as the precursor to (-)-citronellol as well. A limonene synthase has similarly not yet been 
identified from this genus, nor has any other gene related to p-menthane metabolism. While 
molecular, localization, and biochemical data aimed at understanding the metabolic sequence 
leading to monoterpenoid formation in this species are currently limited, the metabolic data 
presented here provide a framework for evaluating the physiological relevance of future cloning, 
expression, and biochemical characterization in this species. Efforts to isolate the corresponding 
transcripts responsible for the metabolism of monoterpenoid volatiles in this species are currently 
underway.  
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Methods and Materials 
 
Unless otherwise specified, all chemical reagents and solvents were purchased from Sigma 
Aldrich Canada Ltd. 
 
Plant materials and growth conditions –Wild-type P. graveolens seeds, P. tomentosum, and the 
Attar of Roses, Mabel Grey, and Graveolens cultivars were obtained from Fibrex Nurseries, Ltd, 
(Stratford-Upon-Avon, UK). P. radens, P. scabrum, P. denticulatum, P. fragrans, P. 
grossularioides, and the Orange Fizz, Coconut, Nutmeg, Apple, and Almond scented geranium 
cultivars were purchased from Richter’s Herbs (Goodwood, Canada). Wild-type P. graveolens 
seeds were germinated in BX M soil (ProMix, Rivière-du-Loup, Canada) supplemented with 
vermiculite under greenhouse conditions at 21 0C with natural lighting. A total of 22 wild-type 
seed grown P. graveolens individuals were obtained. All plant lines were propagated by cuttings 
at 6 week intervals and grown at 24 - 26 0C under neutral photoperiod with natural and 
supplemental lighting in the range of 200-400 mol photons ∙m-1∙s-1(or photosynthetically 
activate radiation, PAR). Nighttime temperatures ranged from 18 - 20 0C. All purpose Miracle 
Gro (NPK 24-8-16) was applied once per week. 
 
Whole plant isotopic labeling – Approximately three weeks after rooting, potted Graveolens 
cultivar or P. graveolens wild-type lines were labeled in a dynamic flow cuvette under 
physiological conditions. Cuttings were rooted in 250 mL pots in BX soil mix and grown in the 
greenhouse for 3-4 more weeks prior to use in labeling experiments. Labeling experiments were 
conducted in a custom built 2 L dynamic flow cuvette into which 4 individual potted plants fit. 
Air flow was maintained at 1.0 L∙min-1 using a pressurized air tank containing normal air (400 
L∙L-1 CO2) for approximately 45 min, during which time gas exchange measurements were 
made with a Li-Cor 840a (Lincoln, Nebraska, USA) CO2/H20 gas analyzer connected to the 
cuvette exhaust. Light intensity, as judged by a Li-Cor 250A quantum meter, was maintained at 
250 PAR using a custom light bank consisting of Cree XPE high intensity white LEDs equipped 
with a potentiometer and high capacity cooling fan (LEDMania, Barcelona, Spain). A mixing fan 
inside the cuvette ensured proper mixing of air within the chamber. The humidity was 
maintained at approximately 70% by passing the air through a wash bottle chilled in an ice bath, 
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and the internal cuvette temperature was kept between 24 - 26 0C based on thermometer 
readings. Once gas exchange readings indicated a near photosynthetic steady state as inferred by 
less than 5% variation in CO2 assimilation over 5 min (generally 45-60 min), the air source was 
switched to an alternate air tank in which the CO2 was 99% enriched by 
13CO2 at the same 
concentration of 400 L∙L-1 CO2 (Linde Canada, Ltd, Mississauga, Ontario, Canada). The decay 
of the 12CO2 signal as detected by the inline IRGA was used to calculate the half life of the 
atmospheric exchange. Labeling times were corrected to the halfway point during introduction of 
the labeling atmosphere into the flow cuvette. Groups of four plants were labeled from 3 – 9 h in 
1 h intervals. Four biological replicates were used for each time point for time course series. At 
the end of labeling experiments, 50 mg of tissue from the youngest 2 leaves were harvested by 
vortexing in 500 µL ethyl acetate containing internal standards as described below. Labeling 
experiments were conducted between 10 am and 2 pm to minimize diurnal effects, except where 
longer labeling experiments precluded this. In that case, experiments were performed from 9 am 
until 6 pm for the longest time points. 
 
Inhibitor treatments –  For exogenous treatments, plants were sprayed at 1, 2, and 3, days before 
labeling. Inhibitor treatments consisted of spraying with one of the following:  150 M 
clomazone, 250 M mevinolin (all in 1% (v/v) dimethylsulfoxide (DMSO)), or 1% DMSO only 
(negative controls)). Inhibitor treated plants were labeled using a single time point of 6 hours. 
For a given labeling experiment of inhibitor treated plants, one plant from each inhibitor group 
and the negative control group were each included. These experiments were carried out over 8 
days for a total of 5 replicates. 
 
Harvest and extraction of plant tissues – Extracts of volatile compounds were obtained from 50 
mg fresh leaf tissue (labeled or unlabeled) steeped overnight at -20 0C in 500 L HPLC-grade 
ethyl acetate (Caledon Laboratory Chemicals) in a 5 mL round bottom screw cap glass extraction 
vial (Fisher Scientific) along with 3,7-dimethyl-1-octanol at 50 g∙mL-1 as internal standard (IS). 
The following day, extraction vials were vortexed at room temperature for 40 min then purified 
over a 0.8 mL glass column containing equal parts silica gel (60 A, 60-100 mesh) and anhydrous 
MgSO4. The column was pre-washed with 400 L ethyl acetate. The sample was applied to the 
column and eluted with an additional 200 L ethyl acetate. Extracts were shielded from light and 
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stored at 4 0C under seal until analyzed by GC-MS. Each wild-type line was sampled and 
extracted on 3 separate occasions. 
 
Volatile analysis by GC-MS – Plant volatiles were analyzed as organic extracts; 1 L purified 
ethyl acetate extracts of plants tissue was analyzed on an Agilent Technologies® 7890B GC 
system coupled to a 5977C mass selective detector utilizing electron impact ionization at 70 eV 
(positive mode). Extracts were analyzed on the following stationary phases: an HP-5ms capillary 
column (30 m length, 0.25 mm i.d., 0.25 m film thickness; Agilent Technologies), an HP-
Innowax (30 m length, 0.25 mm i.d., 0.15 m film thickness; Agilent Technologies), and a 
Cyclodex-B column (30 m length, 0.25 mm i.d., 0.25 m film thickness; Agilent Technologies). 
The HP-5ms column was used for P. graveolens chemotype volatile profiling, while the Innowax 
column was used for label incorporation studies and the -cyclodex column was used for chiral 
GC-MS analysis of limonene enantiomers. For analysis on the HP-5ms column, 1 L extract was 
injected in split mode (1:20) with the injection port set to 225 0C. The oven conditions were as 
follows: 70 0C for 2 min then increasing at 0.7 0C min-1 to 100 0C and held for 3 min, 50 0C min-1 
to 280 0C, with a 5 min final hold time. For analysis on the Innowax column, 1 L extract was 
injected in split mode (1:50) with the injection port set to 225 0C. The oven conditions were as 
follows: 40 0C for 4 min then increasing at 3 0C min-1 to 170 0C, 50 0C min-1 to 260 0C with a 5 
min final hold time. Lastly, for the -cyclodex column, 1 L extract was injected in split mode 
(1:20) with the injection port set to 250 0C. The oven conditions were as follows: 70 0C for 20 
min then increasing at 10 0C min-1 to 210 0C with a 5 min final hold time. For solid phase 
microextraction (SPME) based injections, 100 mg fresh tissue was enclosed in a headspace vial 
and incubated with an exposed polydimethylsiloxane fiber at 22 0C for 60 sec and then injected 
manually with all other settings as above. 
Mass data were collected according to two acquisition regimes. First, data were acquired 
in scan mode (m/z 50-225) with a scan rate of 5 Hz. Second, labeled tissue extracts were re-
analyzed using a selected ion mode (SIM) method that detects exclusively the molecular ion and 
13C labeled isotopologs of prominent volatile compounds. To accomplish this, multiple 
scheduled SIM windows were implemented to monitor label incorporation in the molecular ion 
cluster for compound classes of similar masses as follows: Olefinic monoterpenes, m/z 136-146 
at 30 ms for each mass unit followed by oxygenated monoterpenes, m/z 152-166 at 21 ms per 
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mass unit; select sesquiterpenes, m/z 204-214 coupled with monoterpene ester components at m/z 
154-164 at 15 ms per mass unit. For chiral GC-MS analysis of labelled samples, 3 SIM windows 
were used: Olefinic monoterpenes, m/z 136-146 at 25 ms per mass unit; oxygenated 
monoterpenes, m/z 152-164 with m/z 57 & 70 for internal standards at 19 ms per mass unit; the 
final window for esters and sesquiterpenes m/z 152-164, 204-206 at 18 ms per mass unit.  
 
Data analysis – For the analysis of GC-MS volatile data, peak integration of total ion 
chromatograms and extraction of mass spectra were accomplished using the Agilent MassHunter 
workstation (version B.07 service pack 2). Identification of features was carried out in 
MassHunter Qualitative Analysis based on spectral matches to the NIST2014 mass spectral 
library and comparison to the literature (Jain et al. 2001; Wang et al. 2014; Shellie and Marriott 
2003; Kulkarni et al. 1998). All identifications were confirmed by matching retention times and 
mass spectra to those of in-house authentic standards except where indicated. For untargeted 
profiling, spectral deconvolution, peak picking, and alignment were performed using multiple 
independent approaches. First, raw data were deconvoluted and aligned with MassHunter 
Profinder (version B.08) using the top 132 volatile features reproducibly detected in 177 
biological replicates of cultivars and wild-type P. graveolens lines. In parallel, a Python script 
was developed to facilitate peak picking from integrated peak data obtained in MassHunter 
Qualitative Analysis. Feature matching was ensured by base peak and retention index alignment.  
Principal component analysis, hierarchical clustering, and correlation analysis of all GC-MS data 
was carried out using the Metaboanalyst online suite of metabolomics analysis tools (Xia et al. 
2015; Chong et al. 2018) and standard R packages (Galili 2015; Galili et al. 2017; Wickham 
2016). Absolute quantification was performed by linear regression to individual external 
standard curves constructed from authentic standards and corrected for recovery of the IS. 
 
Supplementary data 
Fig. S1. Leaf morphology of chemotypes 
Fig. S2. Time course labelling time course of monoterpene volatiles in P. graveolens chemotypes 
Fig. S3. SPME-GCMS analysis of p-menthane volatiles from peppermint and the isomenthone 
rich chemotype of P. graveolens 
 
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Competing interest statement 
The authors declare no competing interests. 
  
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Acknowledgements 
This work was supported by a Discovery grant provided by the Natural Sciences and 
Engineering Research Council (RGPIN-2017-06400, to M. P.) and by a John Evans Leadership 
Fund grant from the Canadian Foundation for Innovation (36131, to M. P.). 
 
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Tables 
 
Table 1. Rates of 13C label incorporation into monoterpenes for three P. graveolens chemotypes, as 
judged by linear regression of time course labeling assays 
 (-)-Limonene (+)-Limonene Isomenthone Citronellol Piperitone Geraniol Citronellal 
Citronellol 
chemotype 
       
Slopea 0 0 217.93 465.01 136.47 0 242.03 
Interceptb 0 0 -368.63 -436.01 -143.56 0 -308.15 
R2 0 0 0.805 0.761 0.712 0 0.746 
(Geraniol 
chemotype 
       
Slopea 0.11 3.50 69.68 1.69 0 380.83 0 
Interceptb 0.78 -7.35 409.91 -2.97 0 515.28 0 
R2 0.035 0.505 0.082 0.091 0 0.130 0 
Isomenthone 
chemotype 
       
Slopea 12.88 71.94 1719 0 848.96 0 0 
Interceptb -26.2 -158.2 -3700 0 1849.2 0 0 
R2 0.871 0.955 0.965 0 0.939 0 0 
 
a: pmol 13C∙mg-1 FW∙h-1. b: pmol 13 C∙mg-1 FW.  
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Fig. legends 
Fig. 1. Chemical structures of essential oil components commonly found in the oil of Mentha sp. 
(boxed) and Pelargonium sp.  
 
Fig. 2. Mixed score and loading plot derived from principal component analysis of wild-type P. 
graveolens volatile profiles. Eighty-nine variables representing EO features (mainly 
monoterpenoids and a limited number of sesquiterpenes) were compared among 22 genetically 
distinct groups representing independent seed grown lines. Ethyl acetate extracts were obtained 
from multiple cuttings from each line (n = 4-6) made at different times of the year. The first two 
components explain 77% and 20% of the variation, respectively. Chemical structures indicate the 
key volatile features most associated with each group: (-)-isomenthone (15 lines), (-)-citronellol 
(5 lines), (-)-citronellal, and geraniol (2 lines). 
 
Fig. 3. A, principal monoterpene volatile composition associated with P. graveolens chemotypes 
by fractional peak area. B, summary of data shown in A as pie charts showing the mean 
proportion of key components in the volatile fraction of each chemotype. The fractional peak 
areas for each sample within a chemotype were included in this analysis (n = 6-18 per line (204 
total)). p-values are based on a Student’s two-tailed t-test: ns >= 0.05, * < 0.05, ** < 0.01, *** < 
0.001, **** < 0.0001. 
 
Fig. 4. A, hierarchical clustering of 22 P. graveolens wild-type lines based on volatile profiles 
consisting of 89 features from untargeted GCMS analysis. All but 37 of these features 
corresponded to monoterpenoids. Clustering was performed using the Ward algorithm and 
separated by Euclidean distance. B, heatmap of the 13 most significant volatile features used in 
the hierarchical clustering of P. graveolens lines. C, corresponding correlation matrix of the 
same features using 204 total samples across all 22 lines based on their Pearson correlation 
coefficients. At least three distinct chemotypes could be distinguished based primarily on the 
abundance of p-menthane and citronelloid monoterpenes. An ‘x’ signifies no statistically 
significant correlation. 
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Fig. 5. Effect of inhibitor treatment on 13C label incorporation into monoterpenoid pools. The 
Graveolens cultivated variety, a hybrid which presents a mixed volatile profile consisting of the 
principal volatiles of the (-)-isomenthone, (-)-citronellol, and geraniol rich lines, was treated 
either with clomazone (a MEP pathway inhibitor), mevinolin (a MVA pathway inhibitor), or a 
control solution for 3 days and then subjected to whole plant 13CO2 labeling assays. Volatile 
terpenes were extracted following 6 hours whole-plant labelling. Percent atom labeling was 
calculated by subtracting natural isotopic abundance values obtained from unlabeled control 
samples. Significant differences were inferred through a two-tailed Student’s t-test (n = 5). p-
values are as follows: ns >= 0.05, * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001. 
 
Fig. 6. Time course labeling of monoterpenoid pools in the Graveolens cultivar. Plants were 
equilibrated under a normal atmosphere consisting of 400 ppm CO2 until gas exchange 
measurements indicated a photosynthetic steady state, then labeled with 400 13CO2 for 3 – 8 h 
prior to harvest and extraction. Absolute label incorporation was calculated as the product of the 
fractional labeling of the metabolite pool and absolute concentration as inferred by internal 
standard-normalized external calibration curves established for each analyte. For comparative 
purposes, all labeling values were converted to pmol of 13C equivalents. Linear regressions of the 
resulting incorporation curves are as follows: geraniol, y = 278x – 51 (R2 = 0.892); (-)-
isomenthone, y = 249x – 35 (R2 = 0.848); (-)-citronellol, y = 56x - 2.58 (R2 = 0.625); (+)-
piperitone, y = 65x – 26 (R2 = 0.895); limonene: 29x - 79 (R2 = 0.757). Error bars signify the 
standard error of 4 biological replicates. 
 
Fig. 7. Stereochemical analysis of 13C label incorporation into limonene enantiomers in p-
menthane monoterpene rich and poor P. graveolens chemotypes. (-)-Isomenthone accumulating 
lines are enriched in a variety of p-menthane monoterpenes, including limonene, while (-)-
citronellol and geraniol rich strains are comparatively depleted in cyclic monoterpenes. A, (+)-
limonene and (-)-limonene were separated on a -cyclodex capillary GC column. An extract 
from peppermint (M. x piperita), a known producer of mostly (-)-limonene, was included as a 
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reference. B, Enantiomeric enrichment of limonene enantiomers among P. graveolens 
chemotypes as well as peppermint (M. x piperita) and lemongrass (C. citratus), a known 
producer of (+)-limonene. C, relative incorporation of 13C into (+)- or (-)-limonene during whole 
plant labeling, as judged by analysis of the molecular ion cluster at m/z 136 (M+0) through m/z 
144 (M+8). Limonene content in the citronellol and geraniol chemotypes is approximately 10-
fold lower compared to isomenthone chemotypes. 
 
Fig. 8. Hierarchical cluster analysis of Pelargonium cultivated varieties and species. A, 
hierarchical clustering of 17 Pelargonium varieties including the 3 P. graveolens chemotypes 
based on volatile profiles consisting of 132 features from untargeted GCMS analysis. Clustering 
was performed using the Ward algorithm and separated by Euclidean distance. B, heatmap of 14 
characteristic volatile features used in the hierarchical clustering of P. graveolens lines. C, 
corresponding correlation matrix of the same features using 177 total samples across all 17 
groups based on their Pearson correlation coefficients. An ‘X’ signifies a lack of statistical 
significance. 
 
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Figure 1. Chemical structures of essential oil 
components commonly found in the oil of 
Mentha sp. (boxed) and Pelargonium sp.
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-1 -0.5 0 0.5 1
-1.0
-0.5
0.0
0.5
1.0
-100
-50
0
50
100
-100 -50 0 50 100
Loadings 1
Lo
ad
in
gs
 2
P
C
 2
 (2
0%
)
PC1 (77%)
Geraniol Rich Chemotype
Isomenthone Rich Chemotype
Citronellol Rich Chemotype
Figure 2. Mixed score and loading plot derived 
from principal component analysis of wild-type 
P. graveolens volatile profiles. Eighty-nine 
variables representing EO features (mainly 
monoterpenoids and a limited number of 
sesquiterpenes) were compared among 22 
genetically distinct groups representing 
independent seed grown lines. Ethyl acetate 
extracts were obtained from multiple cuttings 
from each line (n = 4-6) made at different times 
of the year. The first two components explain 
77% and 20% of the variation, respectively. 
Chemical structures indicate the key volatile 
features most associated with each group: (-)-
isomenthone (15 lines), (-)-citronellol (5 lines), (-
)-citronellal, and geraniol (2 lines).
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Isomenthone Geraniol
Citronellol Citronellal
Monoterpene olefins Monoterpene esters
Other monoterpenes
A
Figure 3. A, principal monoterpene volatile 
composition associated with P. graveolens
chemotypes by fractional peak area of all volatile 
compounds. Box plots for (‐)‐isomenthone (I), 
geraniol (G), (‐)‐citronellol (C) chemotypes as well as 
the Graveolens cultivar (Grav.) are shown. B, 
summary of monoterpene content from A as pie 
charts showing the mean proportion of key 
components in the monoterpenoid fraction of each 
chemotype. The fractional peak areas for each 
sample within a chemotype were included in this 
analysis (n = 6‐18 per line (204 total)). p‐values are 
based on a Student’s two‐tailed t‐test: ns >= 0.05, * 
< 0.05, ** < 0.01, *** < 0.001, **** < 0.0001.
80%
14%
2%4%
Isomenthone Chemotype
17%
55%
12%
1%
3%
10% 3%
Geraniol Chemotype
24%
39%
18%
5%
9%
6%
Citronellol Chemotype
11%
74%
11%
2% 3%
Graveolens CV
Fr
ac
tio
n 
of
 to
ta
l p
ea
k 
ar
ea
Group
Grave s CV Isomentho c emotype
Geraniol chemotype Citronellol chemotype
B
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Figure 4. A, hierarchical clustering of 22 
P. graveolens wild‐type lines based on 
volatile profiles consisting of 89 features
from untargeted GCMS analysis. All but 
of 37 of these features corresponded to 
monoterpenoids. Clustering was 
performed using the Ward algorithm 
and separated by Euclidean distance. B, 
heatmap of 13 characteristic volatile 
features used in the hierarchical 
clustering of P. graveolens lines. C, 
corresponding correlation matrix of the 
same features using 204 total samples 
across all 22 lines based on their 
Pearson correlation coefficients. At least
three distinct chemotypes could be 
distinguished based primarily on the 
abundance of p‐menthane and 
citronelloid monoterpenes. An ‘x’ 
signifies no statistically significant 
correlation.
-0.5
-1.0
0.0
0.5
1.0
0-
1-
2-
3-
4-A
B
C
P
g
32
P
g
19
P
g
7
P
g
13
P
g
33
P
g
3
P
g
27
P
g
31
P
g
10
P
g
14
P
g
2
P
g
23
P
g
1
P
g
5
P
g
8
P
g
9
P
g
15
P
g
6
P
g
12
P
g
28
P
g
4
P
g
30
Independent seed grown lines
Eu
cl
id
ea
n 
di
st
an
ce
P
g
32
P
g
19
P
g
7
P
g
13
P
g
33
P
g
3
P
g
27
P
g
31
P
g
10
P
g
14
P
g
2
P
g
23
P
g
1
P
g
5
P
g
8
P
g
9
P
g
15
P
g
6
P
g
12
P
g
28
P
g
4
P
g
30
Row Z-Score
Limonene
Geranial
Piperitone
Citronellyl esters
Limonene
Menthone
Geranyl esters
Citronellic acid
Citronellal
Olefinic monoterpenes
Sesquiterpenes
Geraniol
Citronellol
Isomenthone
Menthone
Sesquiterpenes
Isomenthone
Olefinic monoterpenes
Piperitone
Citronellic acid
Geranial
Citronellol
Citronellyl esters
Citronellal
Geraniol
Geranyl esters
Li
m
on
en
e
M
en
th
on
e
S
es
qu
ite
rp
en
es
Is
om
en
th
on
e
O
le
fin
ic
m
on
ot
er
pe
ne
s
P
ip
er
ito
ne
C
itr
on
el
lic
 a
ci
d
G
er
an
ia
l
C
itr
on
el
lo
l
C
itr
on
el
ly
le
st
er
s
C
itr
on
el
la
l
G
er
an
io
l
G
er
an
yl
 e
st
er
s
Corr.
-4
-2
0
2
4
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Figure 5. Effect of inhibitor treatment on 13C label 
incorporation into monoterpenoid pools. The 
Graveolens cultivated variety, a hybrid which 
presents a mixed volatile profile consisting of the 
principal volatiles of the (‐)‐isomenthone, (‐)‐
citronellol, and geraniol rich lines, was treated 
either with clomazone (a MEP pathway inhibitor), 
mevastatin (a MVA pathway inhibitor), or a control 
solution for 3 days and then subjected to whole 
plant 13CO2 labeling assays. Volatile terpenes were 
extracted following 6 hours whole‐plant labelling. 
Percent atom labeling was calculated by subtracting 
natural isotopic abundance values obtained from 
unlabeled control samples. Significant differences 
were inferred through a two‐tailed Student’s t‐test 
(n = 5). p‐values are as follows: ns >= 0.05, * < 0.05, 
** < 0.01, *** < 0.001, **** < 0.0001.
0.00%
0.25%
0.50%
0.75%
1.00%
%
 A
to
m
 L
ab
el
lin
g
Control Clomazone Mevinolin
***
ns
*** *
*
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Figure 6. Time course labeling of monoterpenoid 
pools in the Graveolens cultivar.  Plants were 
equilibrated under a normal atmosphere consisting 
of 400 ppm CO2 until gas exchange measurements 
indicated a photosynthetic steady state, then 
labeled with 400 13CO2 for 3 – 8 h prior to harvest 
and extraction. Absolute label incorporation was 
calculated as the product of the fractional labeling 
of the metabolite pool and absolute concentration 
as inferred by internal standard‐normalized external 
calibration curves established for each analyte. For 
comparative purposes, all labeling values were 
converted to pmol of 13C equivalents. Linear 
regressions of the resulting incorporation curves are 
as follows: geraniol, y = 278x – 51 (R2 = 0.861); (‐)‐
isomenthone, y = 249x – 35 (R2 = 0.890), (+)‐
piperitone: 65x – 1 (R2 = 0.922), limonene: 28x + 7 
(R2 = 0.444). Error bars signify the standard error of 
5 replicates.
0
500
1000
1500
2000
2500
3000
0 2 4 6 8 10
pm
ol
 13
C
·m
g-
1
FW
Labelling Time (h)
Isomenthone
Piperitone
Citronellol
Geraniol
Limonene
Series8
Series9
Series10
Series11
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Figure 7. Stereochemical analysis of 13C label 
incorporation into limonene enantiomers in p‐
menthane monoterpene rich and poor P. graveolens
chemotypes. (‐)‐Isomenthone accumulating lines 
are enriched in a variety of p‐menthane
monoterpenes, including limonene, while (‐)‐
citronellol and geraniol rich strains are 
comparatively depleted in cyclic monoterpenes. A, 
(+)‐limonene and (‐)‐limonene were separated on a 
b‐cyclodex capillary GC column. An extract from 
peppermint (M. x piperita), a known producer of 
mostly (‐)‐limonene, was included as a reference. B, 
Enantiomeric enrichment of limonene enantiomers 
among P. graveolens chemotypes as well as 
peppermint (M. x piperita) and lemongrass (C. 
citratus), a known producer of (+)‐limonene. C, 
relative incorporation of 13C into (+)‐ or (‐)‐limonene 
during whole plant labeling, as judged by analysis of 
the molecular ion cluster at m/z 136 (M+0) through 
m/z 144 (M+8). Limonene content in the citronellol
and geraniol chemotypes is approximately 10‐fold 
lower compared to isomenthone chemotypes.
34%
37%
63%
3%
88%
0%
25%
50%
75%
100%
C
itr
on
el
lo
l r
ic
h
ch
em
ot
yp
e
G
er
an
io
l r
ic
h
ch
em
ot
yp
e
Is
om
en
th
on
e 
ric
h
ch
em
ot
yp
e
M
en
th
a 
pi
pe
rit
a
C
ym
bo
po
go
n
ci
tra
tu
s
%
(+
)-L
im
on
en
e
16.0 16.5 17.0 17.5 18.0
D
et
ec
to
r R
es
po
ns
e
Time (min)
Mentha piperita
P. graveolens (Isomenthone Rich Chemotype)
100%
32%
19%
0%
68% 81%
0%
25%
50%
75%
100%
C
itr
on
el
lo
l r
ic
h
ch
em
ot
yp
e
G
er
an
io
l r
ic
h
ch
em
ot
yp
e
Is
om
en
th
on
e 
ric
h
ch
em
ot
yp
e
%
 o
f l
ab
el
le
d 
lim
on
en
e
A
B
C
(-)-Limonene (+)-Limonene
(+)-Limonene
(-)-Limonene
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Figure 8. Hierarchical cluster analysis of Pelargonium 
cultivated varieties and species. A, hierarchical 
clustering of 17 Pelargonium varieties including the 
3 P. graveolens chemotypes based on volatile 
profiles consisting of 132 features from untargeted 
GCMS analysis. Clustering was performed using the 
Ward algorithm and separated by Euclidean 
distance. B, heatmap of 14 characteristic volatile 
features used in the hierarchical clustering of P. 
graveolens lines. C, corresponding correlation 
matrix of the same features using 177 total samples 
across all 17 groups based on their Pearson 
correlation coefficients. An ‘X’ signifies a lack of 
statistical significance.
A
E
uc
lid
ea
n 
D
is
ta
nc
e
0-
1-
M
ab
le
 G
re
y 
C
V
O
ra
ng
e 
Fi
zz
 C
V
A
pp
le
 C
V
N
ut
m
eg
 C
V
P
. g
ro
ss
ul
ar
io
id
es
P
. x
9e
fra
gr
an
s
P
. s
ca
br
um
A
lm
on
d 
C
V
P
. d
en
tic
ul
at
um
G
ra
ve
ol
en
s 
C
V
P
. g
ra
ve
ol
en
s 
(G
)
P
ep
pe
rm
in
t C
V
P
. t
om
en
to
su
m
P
. g
ra
ve
ol
en
s 
(I)
A
tta
r o
f R
os
es
 C
V
P
. r
ad
en
s
P
. g
ra
ve
ol
en
s
(C
)
P
. g
ro
ss
ul
ar
io
id
es
A
lm
on
d 
C
V
P
. x
9e
 fr
ag
ra
ns
A
pp
le
 C
V
P
. s
ca
br
um
N
ut
m
eg
 C
V
P
. d
en
tic
ul
at
um
M
ab
le
 G
re
y 
C
V
O
ra
ng
e 
Fi
zz
 C
V
G
ra
ve
ol
en
s 
C
V
P
. G
ra
ve
ol
en
s
(G
)
P
ep
pe
rm
in
t C
V
P
. t
om
en
to
su
m
P
. G
ra
ve
ol
en
s 
(I)
A
tta
r o
f R
os
es
 C
V
P
. r
ad
en
s
P
. G
ra
ve
ol
en
s 
(C
)
B
Row Z-Score
-4
-2
0
2
4
Isomenthone
Geranyl formate
Other
Monoterpenes
Menthone
Citronellyl formate
Geraniol
Piperitol
Geranial
Piperitone
Citronellol
Citronellal
Neral
C
Geranial
Neral
Geranyl formate
Citronellyl formate
Other
Geraniol
Citronellal
Citronellol
Menthone
Piperitol
Isomenthone
Piperitone
Limonene
Monoterpenes
G
er
an
ia
l
N
er
al
G
er
an
yl
 fo
rm
at
e
C
itr
on
el
ly
lf
or
m
at
e
O
th
er
G
er
an
io
l
C
itr
on
el
la
l
C
itr
on
el
lo
l
M
en
th
on
e
P
ip
er
ito
l
Is
om
en
th
on
e
P
ip
er
ito
ne
Li
m
on
en
e
M
on
ot
er
pe
ne
s
Pelargonium species or variety
-0.5
-1.0
0.0
0.5
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