Md. Emran Khan Chowdhury, Bosung Choi, Byoung-Kwan Cho, Joon Bum Kim, Sang Un Park, Savithiry Natarajan, Hyoun-Sub Lim,Hanhong Bae
Abstract
We cloned the full length 4CL ortholog encoding 4-coumarate:coenzymeA ligase from kenaf (Hibiscus cannabiuns, GenBank Accession No. JX548316) using degenerate primers and RACE (rapid amplification of cDNA ends) method. The 4CL is a key regulatory enzyme of the phenylpropanoid pathway that regulates the activation of cinnamic acid, leading to the synthesis of flavonoids and lignin. The 1,704-bp full length of 4CL ortholog had a 1,623-bp open reading frame (ORF) encoding a predicted protein of 540 amino acids. The predicted molecular weight and isoelectric point (pI) of the deduced protein was 59.56 kDa and 6.58,
respectively. The sequence of the deduced amino acid shared 57-79% identities with other 4CL sequences. 4CL ortholog had two conserved putative AMP (adenosine monophosphate)-binding motifs, the SSGTTGLPKGV and GEICIRG domains. A BlastP analysis showed that kenaf 4CL ortholog showed 79% identity with ri4CL2 of Rubus idaeus (AAF91309), which is a class I 4CL involved in lignin synthesis. 4CL ortholog showed differential expression in all tissues during the developmental stages and was highly expressed in stem and root tissues. However, the lowest expression of 4CL ortholog was observed in leaf and mature flower
tissues. 4CL ortholog was responsive to various stress conditions in the stem tissues of 3-week-old kenaf plants. Wounding caused biphasic expression at 6 h and 24 h after treatment. Taken together, the results of this study contribute to the knowledge of the presence of 4CL ortholog and its possible role in lignin biosynthesis, as well as its differential expression during developmental stages.
Keywords: kenaf (Hibiscus cannabinusL.), 4-coumarate: coenzymeA ligase (4CL), lignin biosynthesis, abiotic stress, quantitative real-time PCR (QPCR).
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