Molecular and Functional Characterization of the Key Proanthocyanidin Pathway Enzymes Anthocyanidin Reductases and Leucoanthocyanidin Reductases in <i>Litchi chinensis</i>

The litchi genome has five anthocyanidin reductase (<i>LcANR</i>) and two leucoanthocyanidin reductase (<i>LcLAR</i>) members. The high expression of <i>LcANR1a/2a</i> and <i>LcLAR1/2</i> 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 <i>K</i>_<i>m</i> of LcLAR1/2 to leucocyanidin, 19 and 34 μM, and the <i>V</i>_<i>max</i>, 7 and 5 nmol min^-1 mg^-1, which are rarely reported for other plants. Overexpression of <i>LcANR1a/2a</i> and <i>LcLAR1/2</i> in <i>Arabidopsis ban</i> mutant recovered EC and CT biosynthesis respectively in the seeds; however, the EC-only recovery by LcANR1a/2a is inconsistent with their <i>in vitro</i> activity, indicating that the ANR/LAR function is dependent on characteristic molecular contexts in plants and correlated to the distinct PA profiles in litchi.