Molecular and Functional Characterization of the Key Proanthocyanidin Pathway Enzymes Anthocyanidin Reductases and Leucoanthocyanidin Reductases in Litchi chinensis.

The litchi genome has five anthocyanidin reductase (LcANR) and two leucoanthocyanidin reductase (LcLAR) members. The high expression of LcANR1a/2a and LcLAR1/2 is significantly positively correlated with the abundant proanthocyanidins and (-)-epicatechin (EC) in the pericarp, leaf, root, etc. The recombinant LcANR1a/2a converts cyanidin to both EC and (+)-catechin (CT) (EC:CT ≈ 1:1) and converts delphindin to (+)-gallocatechin and (-)-epigallocatechin; the recombinant LcLAR1/2 converts leucocyanidin to CT. The enzymatic kinetics of the four enzymes are presented, with the respective Km of LcLAR1/2 to leucocyanidin, 19 and 34 μM, and the Vmax, 7 and 5 nmol min-1 mg-1, which are rarely reported for other plants. Overexpression of LcANR1a/2a and LcLAR1/2 in Arabidopsis ban mutant recovered EC and CT biosynthesis respectively in the seeds; however, the EC-only recovery by LcANR1a/2a is inconsistent with their in vitro activity, indicating that the ANR/LAR function is dependent on characteristic molecular contexts in plants and correlated to the distinct PA profiles in litchi.