p-Coumarate:CoA ligase as a key gene in the yield of catechins in tea [Camellia sinensis (L.) O. Kuntze]
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Tea is an important crop known for its beverage and antioxidant polyphenols—catechins and its derivatives. Catechins are synthesized through flavonoid (FL) pathway and stored in the vacuole. A metabolic flux for the operation of FL pathway is maintained through the supply of 4-coumaroyl-CoA of phenylpropanoid pathway. 4-Coumaroyl-CoA is synthesized through the catalytic activity of p-coumarate:CoA ligase (4CL) using 4-coumaric acid and acetyl-CoA as the substrates. The present manuscript reports the full-length cDNA cloning of 4CL from tea (Cs4CL accession number DQ194356) and its association with catechin yield. Cs4CL comprised of 2,165 bp with an open reading frame (ORF) of 1,764 nt, starting from 118 to 1,882 encoding 588 amino acids. Altering catechin content through a variety of environmental conditions such as drought stress (DS), abscisic acid (ABA) and gibberellic acid (GA3) treatments, and wounding established a strong positive correlation coefficient between catechins content and the expression of Cs4Cl. In addition, tea clones with high levels of catechins had higher expression of Cs4Cl whereas tea clones with lower catechins exhibited lower expression of this gene. Exposure of tea shoots to 50–100 μM catechins led to down-regulation of the expression of Cs4CL suggesting product-mediated feedback regulation and an important role for the phenylpropanoid pathway in determining catechin yield in tea.
Keywords4-Coumarate:CoA ligase Catechin Flavonoids Phenylpropanoid Tea
4-coumarate CoA ligase
rapid amplification of cDNA ends
polymerase chain reaction
Authors are thankful to the Council of Scientific and Industrial Research (CSIR), India, for funding the present research work under the New Millennium Indian Technology Leadership Initiative (NMITLI) program entitled “Using functional genomics in plants: development and use of technologies for gene discovery and expression modulation—niche pathway engineering in tea”. KS and AR are grateful to CSIR for awarding Junior/Senior Research Fellowships. PS thanks CSIR for award of project assistantship. Technical help provided by Mr. Digvijay Singh in gene sequencing is duly acknowledged.
- Brignolas F, Lacroix B, Lieutier F, Sauvard D, Drouet A, Claudot AC, Yart A, Berryman AA, Christiansen E (1995) Induced responses in phenolic metabolism in two Norway spruce clones after wounding and inoculations with Ophiostoma polonicum, a bark beetle-associated fungus. Plant Physiol 109:821–827PubMedGoogle Scholar
- Diatchenko L, Lau Y-FC, Lukyanov K, Gurskay N, Sverdlov ED (1996) Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Biochem 93:6025–6030Google Scholar
- Fischer TC, Halbwirth H, Roemmelt S, Sabatini E, Schlangen K, Andreotti C, Spinelli F, Costa G, Forkmann G, Treutter D, Stich K (2006) Induction of polyphenol gene expression in apple (Malus × domestica) after the application of a dioxygenase inhibitor. Physiol Plant 128:604–617CrossRefGoogle Scholar
- Ghawana S, Singh K, Raizada J, Rani A, Bhardwaj PK, Kumar S (2004) A method for rapid isolation of RNA and a kit thereof. A joint patent between CSIR and DBT 0344 NF 2004/IN Dated 30.03.06 (filed)Google Scholar
- Singh K (2006) Cloning and characterization of gene(s) involved in catechin biosynthesis in tea (Camellia sinensis (L.) O Kuntze). Ph.D. Dissertation, Guru Nanak Dev University, Amritsar, IndiaGoogle Scholar
- Singh K, Raizada J, Bhardwaj P, Ghawana S, Rani A, Singh H, Kaul K, Kumar S (2004) 26S rRNA-based internal control gene primer pair for reverse transcription-polymerase chain reaction-based quantitative expression studies in diverse plant species. Anal Biochem 335:330–333PubMedCrossRefGoogle Scholar
- Singh K, Kumar S, Rani A, Gulati A, Ahuja PS (2008a) Phenylalanine ammonia-lyase (PAL) and cinnamate 4-hydroxylase (C4H) and catechins (flavan 3-ols) accumulation in tea. Funct Integ Genom. doi: 10.1007/s10142-008-0092-9