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.