Functional Characterization of Proanthocyanidin Pathway
Enzymes from Tea and Their Application for
Metabolic Engineeringl[W][OA]
Yongzhen Pang2, I. Sarath B. Abeysinghe, Ji He, Xianzhi He, David Huhman, K. Mudith Mewan,
Lloyd W. Sumner, Jianfei Yun, and Richard A. Dixon3*
Plant Biology Division, Samuel Roberts Noble Foundation, Ardmore, Oklahoma 73401 (Y.P., J.H., X.H., D.H.,
L.W.S., J.Y., R.A.D.); and Biochemistry Division, Tea Research Institute of Sri Lanka, Talawakelle 22100,
Sri Lanka (I.S.B.A., K.M.M.)
Tea (Camellia sinensis) is rich in specialized metabolites, especially polyphenolic proanthocyanidins (PAs) and their precursors.
To better understand the PA pathway in tea, we generated a complementary DNA library from leaf tissue of the blister blight-
resistant tea cultivar TRI2043 and functionally characterized key enzymes responsible for the biosynthesis of PA precursors.
Structural genes encoding enzymes involved in the general phenylpropanoid/flavonoid pathway and the PA-specific branch
pathway were well represented in the library. Recombinant tea leucoanthocyanidin reductase (CsLAR) expressed in Escherichia
coli was active with leucocyanidin as substrate to produce the 2R,3S-trans-flavan-ol (+)-catechin in vitro. Two genes encoding
anthocyanidin reductase, CsANR1 and CsANR2, were also expressed in E. coli, and the recombinant proteins exhibited similar
kinetic properties. Both converted cyanidin to a mixture of (+)-epicatechin and (-)-catechin, although in different proportions,
indicating that both enzymes possess epimerase activity. These epimers were unexpected based on the belief that tea PAs are
made from (-)-epicatechin and (+)-catechin. Ectopic expression of CsANR2 or CsLAR led to the accumulation of low levels of PA
precursors and their conjugates in Medicago truncatula hairy roots and anthocyanin-overproducing tobacco (Nicotiana tabacum),
but levels of oligomeric PAs were very low. Surprisingly, the expression of CsLAR in tobacco overproducing anthocyanin led to
the accumulation of higher levels of epicatechin and its glucoside than of catechin, again highlighting the potential importance of
epimerization in flavan-3-ol biosynthesis. These data provide a resource for understanding tea PA biosynthesis and tools for the
bioengineering of flavanols.
Tea (Camellia sinensis) originated in southern China.
It is the most widely produced and consumed bever-
age in the world after water and has received a great
deal of attention with respect to its numerous health
benefits to humans (Lin et al., 2003). Although the
composition of tea varies with cultivar, age of leaf,
season, and processing method (Lin et al., 1996), tea
contains uniquely high concentrations of polyphenolic
compounds, which consist mainly of six flavan-3-ols:
epigallocatechin gallate (EGCG; 9%-13% of dry
1 This work was supported by the National Science Foundation
program for Developing Country Collaborations in Plant Genome
Research (grant no. 04-23), the Samuel Roberts Noble Foundation,
and Forage Genetics International.
2 Present address: Institute of Botany, Chinese Academy of
Sciences, Beijing 100093, People's Republic of China.
3 Present address: Department of Biological Sciences, University of
North Texas, Denton, TX 76203.
* Corresponding author; e-mail Richard.Dixon@unt.edu.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy de-
scribed in the Instructions for Authors (www.plantphysiol.org) is:
Richard A. Dixon (Richard.Dixon@unt.edu).
[W] The online version of this article contains Web-only data.
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www.plantphysiol.org/cgi/doi/10.1104/pp.112.212050
weight), epigallocatechin (EGC; 3%-6%), epicatechin
gallate (ECG; 3%-6%), epicatechin (1%-3%), galloca-
techin (GC; 1%-2%), and catechin (1%-2%; Balentine
et al., 1997; Fig. 1); together, these can constitute more
than 30% of the dry weight. A further 3% of the dry
weight is composed of proanthocyanidins (PAs; olig-
omeric or polymeric flavan-3-ols).
Increasing evidence suggests that tea flavan-3-ols
are beneficial for human health as a result of their
antioxidant capacity (Luximon-Ramma et al., 2006),
chemopreventive activities against prostate and ovar-
ian cancers (Bemis et al., 2006; Ravindranath et al.,
2006), antiobesity and antidiabetic effects (Yang and
Koo, 2000; Kao et al., 2006; Wolfram et al., 2006), and
cardiovascular disease prevention properties (Yang
and Koo, 2000). Tea flavanols, especially catechins,
may also play roles in the defense of tea against
infection/infestation (Punyasiri et al., 2004).
The blister blight disease caused by the fungal
pathogen Exobasidium vexans is the major disease af-
fecting tea quality and yield in Asia (Arulpragsam,
1992). Previous investigations have indicated that
flavan-3-ol content and composition differ in different
blister blight-resistant and -susceptible tea cultivars
and at different stages during infection. The levels of
(-)-E (for epicatechin) in resistant cultivars are
significantly higher than in susceptible ones, whereas
Plant Physiology, March 2013, Vol. 161, pp. 1103-1116, www.plantphysiol.org 2013 American Society of Plant Biologists. All Rights Reserved. 1103
