Molecular and Functional\nCharacterization of the Key\nProanthocyanidin Pathway Enzymes Anthocyanidin Reductases and Leucoanthocyanidin\nReductases in <i>Litchi chinensis</i>

The litchi genome has five anthocyanidin reductase (<i>LcANR</i>) and two leucoanthocyanidin reductase (<i>LcLAR</i>) members.\nThe high expression of <i>LcANR1a/2a</i> and <i>LcLAR1/2</i> is significantly positively correlated with the abundant proanthocyanidins\nand (−)-epicatechin (EC) in the pericarp, leaf, root, etc.\nThe recombinant LcANR1a/2a converts cyanidin to both EC and (+)-catechin\n(CT) (EC:CT ≈ 1:1) and converts delphindin to (+)-gallocatechin\nand (−)-epigallocatechin; the recombinant LcLAR1/2 converts\nleucocyanidin to CT. The enzymatic kinetics of the four enzymes are\npresented, with the respective <i>K</i><sub><i>m</i></sub> of LcLAR1/2 to leucocyanidin, 19 and 34 μM, and the <i>V</i><sub><i>max</i></sub>, 7 and 5 nmol min<sup>–1</sup> mg<sup>–1</sup>, which are rarely reported for other plants.\nOverexpression of <i>LcANR1a/2a</i> and <i>LcLAR1/2</i> in <i>Arabidopsis ban</i> mutant recovered EC and CT biosynthesis\nrespectively in the seeds; however, the EC-only recovery by LcANR1a/2a\nis inconsistent with their <i>in vitro</i> activity, indicating\nthat the ANR/LAR function is dependent on characteristic molecular\ncontexts in plants and correlated to the distinct PA profiles in litchi.