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.