DISCUSSION
Benzenoid and terpenoid volatiles dominate the floral scent note in
summer-blooming J. auriculatum (Bera et al., 2015). The present
study suggest that contents of the major scent compounds were high at
Stages 4 and 5 when the flowers start unfurling and becomes fully open
under in situ conditions. The sampling time for S4 was between
5-6 pm in the evening and S5 was between 8-9 pm at night. These results
agreed well with our recent finding where highest temporal emission was
between 6-10 pm at night, where sampling was done at 4 h interval
(Barman and Mitra., 2019). The findings of our result also correspond
well with the findings of other summer jasmines where the maximum
emission was observed at late night on the same day of flower blooming
(Bera et al., 2017; Pragadheesh et al., 2017; Yu et al., 2017). These
findings show that this species is nocturnally active to attract night
pollinators (Paul et al., 2019).
In order to understand the metabolism of major emitted volatiles
comprising benzenoids (benzyl acetate, 2-phenylethanol and benzyl
alcohol) and terpenoids (linalool, α-farnesene and farnesol), an attempt
was made to study the pathway enzymes and genes which contribute to the
biosynthesis of these volatile compounds at different floral maturation
stages. The biosynthesis and emission of volatiles from J.
auriculatum flowers showed a floral growth and maturation specific
pattern. The enzyme PAL is generally known as the rate limiting enzyme
in the benzenoid/phenylpropanoid biosynthesis pathway. The gene
expression and enzyme activity levels of PAL correlated well with the
benzenoids emission. A similar observation in the enhanced rate of PAL
expression and activity was also observed in petunia and tuberose that
correlated with their benzenoids emission (Cheng et al., 2016; Maiti and
Mitra, 2017). The gene expression level and activity of BEAT, the
ultimate enzyme for benzyl acetate formation was high at flower blooming
stage. This result suggest that highest rate of biosynthesis takes place
when flower starts blooming and the compound formed is present as inter
pool in the floral tissue with some basal emission at this stage and
maximum emission occurs when flower was fully bloomed. The compound
benzyl acetate is reported to be emitted in higher rate at night to
attract nocturnal pollinators (Dudareva et al., 1998). The variations
observed in BEAT enzyme activity at different stages of petal growth and
maturation are similar to those observed by Bera et al. (2017) inJ. sambac where BEAT activity reached its peak on the same day of
maximum emission. Shalit et al. (2003) has also reported a maximal
activity of acetyl-transferase at a stage prior to the stage where
maximum acetate ester volatile emission was observed from petals of
rose. The higher enzyme activity of PAR and MTS at stage 3 indicated
that biosynthesis of 2-phenylethanol and linalool was maximum at the bud
stage where no emission of these compounds was detected; however high
emission of 2-phenylethanol and linalool was detected when the flower
started blooming. It was reported earlier that these compounds were
present as glycosyl-linked volatile in floral tissue (Watanabe et al.,
1993; Oka et al., 1999). It can be inferred that these compounds after
biosynthesis was glycosylated prior to their storage in floral tissue,
and upon flower opening emission of these de-glycosylated volatiles
occurs. PAR activity was highest at mature bud stage and a high
abundance of this gene occurred throughout the floral lifespan unlike
the results obtained from enzyme activity. The plausible reason might be
that the transcript accumulation is active at different stages of petal
growth and maturity, but due to lack of substrates the enzyme activity
was limited to late bud stage. Gene expression levels of JaMTSalso correlated well with the activities of its corresponding enzyme. We
reported earlier that higher content of glycosylated volatiles was
present in mature buds of J. auriculatum (Barman and Mitra,
2019); in this study we found that upon flower opening the stored
glycosylated volatiles get released as free volatiles by the action of
β-glucosidase as evidenced by enhanced transcript levels and uplifted
β-glucosidase enzyme activity. This suggests that β-glucosidase plays an
important role in scent production by hydrolysis of glycosyl moiety
attached to volatile molecule in J. auriculatum . Thus, upon
flower opening the scent emission is enhanced due to increased
hydrolytic activity. Similar observation was reported fromNarcissus flowers where β-glucosidase activity correlated with
the increase in scent emission by cleaving the glucosyl bound volatiles
from open flowers (Reuveni et al., 1999).
Higher expressions of JaHMGS and JaHMGR at stage 2 and
stage 3 indicated that these upstream genes start expressing at bud
stages for formation of precursors for sesquiterpene volatiles
biosynthesis. A similar observation in other scented Jasminumspecies has been reported earlier (Yu et al., 2017). Higher expression
levels of JaMYB at stage 1 indicates that these TFs has a role in
regulation of upstream genes responsible for phenylpropanoid
biosynthesis at early bud stage. This TF belonging to R2R3 family
upregulates the early steps of phenylpropanoid/benzenoid biosynthesis
(Liu et al., 2015). R2R3 TFs has also been reported to enhance aroma
production by regulating the biosynthesis of phenylpropanoids in tomato,
maize and other scented flowering plants (Jian et al., 2019; Liu et al.,
2015; Ramya et al., 2018).
Apart from different developmental, temporal and spatial patterns,
different environmental factors also influence the accumulation of
specialized volatile metabolites in plants (Cheng et al., 2016). In the
past decades the impact of varying air temperature out of all other
environmental factors has been reported to have significant influence on
the scent emission from many commercially important flowers (Hu et al.,
2013; Cna’ani et al., 2015; Zeng
et al., 2019). However, no detailed reports are available on the
emission of volatiles from any Jasminum species, a commercially
important flowering species in tropical countries. In the present study
it was observed that the contents of most of the compounds comprising
benzenoid and terpenoid volatiles was high either at 25°C or 30°C. At
20°C and 35°C, air temperatures, much lower contents of emitted and
endogenous volatiles were recorded. The decrease in both emitted and
endogenous floral volatile contents at the two border-range air
temperatures (set out for conducting growth chamber experiments)
conditions indicates that ambient temperature affects both metabolism
and vaporization of the compounds. A schematic diagram in Figure
9 provides holistic overview about the influence of different air
temperatures on emission of major floral volatiles in J.
auriculatum . The release of scent volatiles from T. repens(Jakobsen and Olsen 1994) and P. hybrida (Sagae et al., 2008)
flowers increased with increase in temperature up to 35°C. It was also
reported that emission of volatiles from P. hybrida flowers was
low at 20°C but with high contents of endogenous volatiles accumulation
at the same temperature (Sagae et al., 2008) unlike the findings
observed by us. The plausible reason for this reduced metabolism inJ. auriculatum flowers at 20°C might be because of its tropical
adaption which made the genetic makeup changed in such a way that would
support the maximum production of floral scent upon growing under a
temperature range of 25°C to 30°C. A recent study conducted on emission
of scent from cut rose revealed that an increased release of benzenoid
volatiles was observed at 30°C unlike the contents observed under 5°C
and 15°C (Zeng et al., 2019).
Environmental conditions not only affect the vaporization of volatile
compounds from flowers, but also the biosynthesis, particularly under
different air temperature regimes (Cheng et al., 2016). In this study we
observed that the contents of both emitted and endogenous volatiles inJ. auriculatum flowers were modulated under temperature regime,
suggesting an important role of temperature on volatiles metabolism. To
establish the hypothesis, the amount of active enzymes in the floral
volatiles pathway was measured in terms of their in vitroactivities from flowers of J. auriculatum growing under different
air temperature regime. Activities of BEAT, PAR, MTS and β-glucosidase
corresponded well with the contents of their respective volatiles as
observed at different stages of floral maturation. Expression analysis
of genes also supported the in vitro activities data. Similar
observations were found with the reduced contents of benzenoid volatiles
in P. hybrida flowers where incubation of this plant at higher
air temperature led to downregulation of scent related genes (Cna’ani et
al., 2015). Downregulation of emitted volatiles originating from
benzenoid/phenylpropanoid pathway was observed when air temperature
reached at 35°C. To understand this phenomenon, we attempted to study
expression pattern and activity of PAL, the first enzyme in the
phenylpropanoid pathway leading to the formation of many phenolic
compounds including benzenoid volatiles and anthocyanins. Reduced enzyme
activity as well as gene expression of PAL at 20°C and 35°C was well
supported by previous finding where too low or too high ambient
temperature hindered the activity of this enzyme (Shaked-Sachray et al.,
2002). The expression levels of most of the intermediate genes
(JaHMGS, JaHMGR ) as well as the JaMYB , R2R3 family
transcription factor also corelated well with the contents of floral
scent under different air temperature condition. Similar findings inP. hybrida and R. hybrida flowers were reported earlier
where too low or too high ambient temperature affected the transcript
levels of intermediate genes responsible for the biosynthesis of
volatile compounds (Cheng et al., 2016; Zeng et al., 2019). Our present
findings suggest that air temperature range of 25°C followed by 30°C
aids in the maximal release of floral scent from J. auriculatum .
The floral volatiles metabolism and emission were shown at first to
increase and then decreased with the enhanced air temperature regime.
Similar observation was also found in Lilium flowers, where
floral scent upsurge with increase in temperature, followed by a gradual
decrease was recorded (Hu et al., 2013). Lower release of volatiles at
20°C might be because of reduced enzyme activity responsible for
biosynthesis of floral scent as was observed in our study. An increased
temperature of 35°C inhibited the expression of pathway genes which
finally resulted in reduced activity of end product enzymes responsible
for the biosynthesis as well as emission of scent volatiles fromJ. auriculatum flowers. Reduced emission of floral scent at
higher temperatures of 35°C was also reported from P. axillarisflowers (Sagae et al., 2008).
Non-volatile metabolites identified from floral extracts comprised
mainly of primary metabolites viz. sugars, organic acids, fatty acids
and a few secondary metabolites like phenolic acids. The higher
abundance of monosaccharides mainly fructose, glucose and arabinose
under 25°C supports well with the findings observed from volatile
contents under different temperature conditions. These monosaccharides
are the ultimate precursors for most of the biosynthetic pathways
involved in the biosynthesis of floral volatiles (Pickersky et al.,
2006). Therefore, enhanced contents of these primary metabolites
indicate not only the downstream precursors (phenolic acids) but also
the upstream precursors (eg. monosaccharides) are affected by variations
in air temperature conditions. Our findings suggest an increased content
of most of the observed sugar alcohols under the upper border-range
temperature of 35°C. This may be due to the fact that at this
temperature the plants might have shifted its carbon pool to synthesize
sugar alcohols rather than biosynthesis of specialized scent molecules
to protect themselves from mild heat stress. It was earlier reported
that sugar alcohols are used as osmoprotectants and usually accumulated
in plants facing many kinds of abiotic stresses (Tarczynski et al.,
1993; Sheveleva et al., 1998). It was also observed that the abundance
of most of the phenolic acids and other intermediate precursors was
highest under the temperature where highest scent emission rate was
observed. Increased amount of these precursors supports the increased
scent compound formation under 25°C and 30°C.