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.