FIGURE CAPTIONS
Figure 1. Emission of
benzenoid (a-c) and terpenoid (d-f) volatiles from J. auriculatumflowers during their lifespan, grown under in situ condition.
Emitted volatiles were measured by headspace collection from intact
flowers at six different floral maturation stages i.e. from green bud to
senescence. Histogram represent the average values of three to five
independent experimental setup where SD is represented in vertical
lines. Data indicated with different alphabets differ significantly
according to Tukey’s HSD (p < 0.05). The collection of
volatiles was carried out in a glass headspace for an hour.
Figure 2. Changes in the
amount of active proteins (in terms of their in vitro activities)
of key enzymes for biosynthesis of
phenylpropanoids/benzenoids/terpenoids volatiles in flowers of J.
auriculatum collected at different floral maturation stages. Levels of
PAL specific activity responsible for biosynthesis oftrans -cinnamic acid at different bud and flowering stages (a).
Levels of β-glucosidase specific activity responsible for hydrolysis of
glycosyl bound volatiles at different bud and flowering stages (b).
Levels of BEAT specific activity responsible for biosynthesis of benzyl
acetate at different bud and flowering stages (c). Levels of PAR
specific activity responsible for biosynthesis of 2-phenylethanol at
different bud and flowering stages (d). Levels of MTS specific activity
responsible for biosynthesis of linalool at different bud and flowering
stages (e). GC chromatogram showing the product formation catalysed by
BEAT (f), PAR (g) and MTS (h) enzymes extracted from flowers of
different developmental stages where product formation and substrate are
marked, IS indicates internal standard used. Histograms represent the
average values of three to five independent experimental setup where SD
is represented in vertical lines. Data indicated with different
alphabets differ significantly according to Tukey’s HSD (p< 0.05).
Figure 3. Expression of
candidate genes of scent
biosynthetic pathway at different floral maturation stages of J.
auriculatum . Agarose gel images of amplified PCR products forJaPAL, JaBEAT, JaPAR, JaMYB, Jaβ-Glu, JaHMGS, JaHMGR, JaMTS (a).
Color codes indicate the relative expression analysis of scent related
genes by semi-quantitative RT-PCR with actin as the internal standard at
six stages of floral maturation, where lower intensity is indicated in
green and higher intensity is indicated in red (b). qPCR analyses ofJaBEAT and JaMTS , the ultimate genes responsible for the
biosynthesis of benzyl acetate and linalool, respectively, the major
scent contributors in J. auriculatum (c). Values are represented
as mean of three individual replicates where vertical lines indicate the
SD. Data indicated with different alphabets differ significantly
according to Tukey’s HSD (p < 0.05).
Figure 4. Altered emission of scent volatiles from J.
auriculatum flowers grown under a
range of fixed temperatures in growth chamber. Plants were
simultaneously grown under four different air temperature conditions
viz. 20, 25, 30 and 35 °C. Emitted
volatiles were collected on the day of blooming from the headspace of
intact flowers collected individually in the evening between 6.00 p.m.
to 7.00 p.m. Values are represented as mean of three individual
experimental setups where vertical lines indicate the SD. Data indicated
with different alphabets differ significantly according to Tukey’s HSD
(p < 0.05).
Figure 5. Variations in the content of internal pool of scent
volatiles in the form of free-endogenous and glycosyl-bound compounds inJ. auriculatum flowers grown under different air temperature
regime. Plants were grown under four different temperature conditions
viz. 20, 25, 30 and 35 °C. Sampling of free endogenous volatiles were
done from these flowers used earlier for collection of emitted
volatiles. Petals were immediately
excised and extracted with dichloromethane for immediate release of the
internal free scent molecules by organic solvent. The sampling of
glycosylated volatiles was carried out from petals of mature buds by
enzymatic treatment for hydrolysis of glycosyl moiety from attached
volatile compound. Values are represented as mean of three individual
replicates where vertical lines indicate the SD. Data indicated with
different alphabets differ significantly according to Tukey’s HSD
(p < 0.05).
Figure 6. Levels of
specific activities of key enzymes responsible for biosynthesis of
phenylpropanoids/ benzenoids/terpenoids in flowers of J.
auriculatum collected from plants growing under a range of air
temperature conditions. Plants
were grown under four different air temperature conditions viz. 20°C,
25°C, 30°C and 35 °C. Levels of PAL specific activity responsible for
biosynthesis of trans -cinnamic acid from flowers kept under
different temperature conditions (a). Levels of β-glucosidase specific
activity responsible for hydrolysis of glycosyl bound volatiles from
flowers kept under different temperature conditions (b).
Levels of BEAT specific activity
responsible for biosynthesis of benzyl acetate from flowers kept under
different temperature conditions (c). Levels of PAR specific activity
responsible for biosynthesis of 2-phenylethanol from flowers kept under
different temperature conditions (d). Levels of MTS specific activity
responsible for biosynthesis of linalool from flowers kept under
different temperature conditions (e). The crude extracts for activity
studies of particular enzymes under different temperature were collected
either at late bud stage or at flower blooming stage depending on the
higher content of particular enzymes as was observed from the in
situ developmental stage study as described in the Results section. GC
chromatogram showing the product formation catalysed by BEAT (f), PAR
(g) and MTS (h) enzymes extracted from flowers collected from different
temperature conditions where product formation and substrate are marked,
IS indicates internal standard used. Graphs represent the average values
of three to five independent experimental setup where SD is represented
in vertical lines. Data indicated with different alphabets differ
significantly according to Tukey’s HSD (p < 0.05).
Figure 7.Expression
of scent related genes in J. auriculatum flowers grown under
a range of fixed air temperatures
successively in growth chambers. Plants were grown under four different
temperature conditions viz. 20°C, 25°C, 30°C and 35°C. Agarose gel
images of amplified PCR product for JaPAL, JaBEAT, JaPAR, JaMYB,
Jaβ-glu, JaHMGS, JaHMGR, JaMTS transcripts in flowers collected from
plants growing under different temperature conditions (a). Colour codes
indicate the relative expression analysis of scent related genes by
semi-quantitative RT-PCR where actin is used as the internal standard,
lower intensity is indicated in green and higher intensity is indicated
in red (b). RT-qPCR analyses of JaBEAT and JaMTStranscripts, the ultimate genes responsible for the biosynthesis of
benzyl acetate and linalool, the major scent contributors in J.
auriculatum (c). Values are represented as mean of three individual
replicates where vertical lines indicate the SD. Data indicated with
different alphabets differ significantly according to Tukey’s HSD
(p < 0.05).
Figure 8. Changes in the
relative contents of non-volatile
metabolites in mature buds ofJ. auriculatum grown under a range of fixed air temperatures.
Plants were grown under four different temperature conditions
viz. 20°C, 25°C, 30°C and 35°C.
Mature buds growing under different temperature conditions were
successively collected and freeze
dried to obtain concentrated amounts of non-volatile metabolites. Colour
code represents the relative abundance of individual compound as was
calculated on the basis of ribitol, the internal standard (a). Light
yellow and dark blue represent lowest and highest relative abundance of
non-volatile metabolites. Values are represented as mean of five
individual replicates. PCA score
plot (b) and loading plot (c) of the non-volatile metabolite compounds
collected from flowers of plants kept under different temperature
conditions. PC1 and PC2 are the first two principal components. The
score plot indicates the clear segregation of each temperature treated
plants. The loading plot indicate the compounds responsible for the
segregation of treatments.
Figure 9. A simplified overview of the pathways involved in
biosynthesis of major volatile compounds in J. auriculatumflowers. The pathway genes and enzymes highlighted in blue are measured
either in terms of the amount of active enzymes and/or gene expression
levels from flowers at appropriate maturation stages (where maximum
scent emission occurred) collected under varying air temperature regime.
Colour code represents the abundance of floral volatiles emitted under
different air temperature conditions where yellow and red represent
lower and higher abundance, respectively. The formation of monoterpene
volatiles take place in the plastid via the MEP pathway. The
biosynthesis of phenylpropanoids/benzenoids and sesquiterpene volatiles
occur in the cytoplasm via shikimate/chorismate and MVA pathways
respectively. The dotted boundary around the phenylpropanoid/benzenoid
biosynthetic pathway indicates that MYB transcription factor has a
potential role in their regulation. Volatile compounds (marked as
droplets) after glycosylation are transported to the vacuole for
storage. Perhaps upon physiological necessity these stored glycosylated
compounds are released as free volatiles by the aid of hydrolytic
enzymes viz. β-glucosidase. Abbreviations: BEAT, acetyl-CoA:benzyl
alcohol acetyltransferase; β-Glu, β-glucosidase; Ery4P,
erythrose-4-phospate; FPP, farnesyl pyrophosphate; GA-3P,
glyceraldehyde-3-phosphate; GPP, geranyl pyrophosphate; HMGR,
hydroxymethylglutaryl-Coenzyme A reductase; HMGS,
hydroxymethylglutaryl-Coenzyme A synthase; IPP, iso-pentenyl
pyrophosphate; LOX, lipoxygenase; MEP, methylerythritol phosphate; MTS,
monoterpene synthase; MVA, mevalonic acid; PAL, phenylalanine ammonia
lyase; PAR, phenylacetaldehyde reductase; PEP, phosphoenolpyruvate; Phe,
L phenylalanine; Pyr, pyruvate.