3.1.3 Expression levels of scent related genes at different floral maturation stages
The levels of transcripts accumulation related to the formation of major scent volatile compounds were studied using semi-quantitative RT-PCR. These include phenylalanine ammonia-lyase (JaPAL ), acetyl coenzyme A: benzyl alcohol acetyl transferase (JaBEAT ) and phenylacetaldehyde reductase (JaPAR ) that are related to the biosynthesis of major benzenoids volatiles, and candidate genes for terpenoid volatiles viz. monoterpene synthase (JaMTS ), 3-hydroxy-3-methylglutaryl coenzyme A synthase (JaHMGS ), 3-hydroxy-3-methylglutaryl coenzyme A synthase (JaHMGR ). FurtherJaMYB , a transcription factor that regulates the early steps of benzenoid/phenylpropanoid biosynthesis, and β-glu cosidase gene (JaβGlu ) that function to de-glycosylate glucosyl-linked volatiles were also studied. The PCR-amplified transcript bands obtained from cDNA of J. auriculatum were sequenced and subsequently blasted to find homologies with the predicted amino acid sequences of the genes available in the public database. The sequence data information for these gene fragments were later submitted to the GenBank databases under accession numbers MT542640, MK879598, MN641835, MK903849, MN641834, MN641833, MK879597 and MK789770 for J. auriculatum respectively.
Transcript abundance of JaPAL and JaBEAT was found to be higher at flower opening (S4) stage. The accumulation of JaPARgene did not vary much within the floral stages apart from a slightly increased abundance at early bud (S1) stage. The levels of JaMYBtranscript accumulation were found to be highest at early bud (S1) stage and gradually declined with the maturity of buds (Figure 3a, b ). In order to understand the biosynthesis of terpenoid volatiles, the transcript levels of JaHMGS , JaHMGR (upstream genes involved in the biosynthesis of sesquiterpene volatiles) andJaMTS (the final gene involved in the biosynthesis of monoterpene linalool) were assessed (Figure 3a, b ). It was observed that the transcript level of JaHMGS was highest at mid-bud (S2) stage while late bud (S3) stage showed highest JaHMGR transcript accumulation; the transcript accumulation of JaMTS increased at late bud (S3) stage. Many volatile compounds are biosynthesized and stored as glycosyl bound form. They are further hydrolyzed by several glycolytic enzymes for easy release of volatile compounds. The transcript levels of β-glucosidase (Jaβ-Glu ) gradually increased from late bud (S3) stage to flower opening (S4) stage and then decreased in the subsequent floral maturation stages.
As volatile compounds namely, benzyl acetate and linalool contribute principally for the signature scent note of J. auriculatum flower (Barman et al., 2020), the relative expression of JaBEAT andJaMTS genes were also monitored using real time quantitative PCR (qRT-PCR). The Ct values obtained from qPCR analysis were utilized for calculating the fold change to understand the relative gene expression level (Figure 3c ). It was found that the relative gene expression at different petal growth and maturation stages correlated well with the intensities of the transcripts observed via semi-quantitative RT-PCR.