Functional Characterization Of Key Enzyme Genes LARs And ANR In Populus Proanthocyanidin Biosysnthesis

The flavonoid-derived proanthocyanidins (PAs, also called condensed tannins) are one class of the major defence phenolics in poplar leaves. Leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR) catalyze the synthesis of catechin and epicatechins, respectively. Catechin and epicatechins as initiating monomers of PA synthesis, from3,4-cis-leucocyanidin and thus is the first committed step in PA biosynthesis. In this study, three full-length cDNAs named PtrLAR1, PtrLAR3and PtrANR were isolated from Populus trichocarpa. Sequence comparison of the PtrLARs and PtrANR with other known proteins reveals high amino acid sequence similarity. The expression profiles of PtrLAR and PtrANR cDNAs were investigated in various tissues of P. trichocarpa. Semi-quantitative reverse-transcription (RT) PCR and quantitative real-time PCR analysis demonstrated that PtrLAR and PtrANR were expressed in various tissues and the highest level of expression was observed in roots. We also determined the content of condensed tannin in different tissues and highest level was observed in roots, consistent to the expression profiles of PtrLAR and PtrANR.To determine their functions, PtrLAR and PtrANR were overexpressed in transgenic Chinese white poplar (P. tomentosa Carr.) plants via Agrobacterium mediation. A significant higher accumulation of condensed tannins was detected in transgenic plants harboring the PtrLAR and PtrANR overexpression construct, compared to the wild-type control. Since biosynthesis of anthocyanin and condensation tannin shares the same precursor. In order to detect whether overexpression of PtrLAR and PtrANR infect anthocyanin biosynthesis in transgenic plants, we determined the anthocyanin content of transgenic plants. The results showed that anthocyanin accumulation was obviously decreased in transgenic plants overexpressed PtrLAR and PtrANR. These findings indicated that PtrLAR1and PtrANR may play an important role in condensed tannin synthesis.In previous studies, it is demonstrated that catechin is derived from leucocyanidin by LAR, while epicatechins are synthesized from anthocyanidins by ANR. To determine the function of PtrLAR1and PtrANR, condensed tannins were extracted from transgenic poplar plants. HPLC analysis showed that PtrANR could increase dramatically epicatechin content, whereas PtrLARl overexpression improved accumulation of both catechin and epicatechin. This result suggested that PtrLAR1might also have catalytic function in biosynthesis of epicatechins in poplar.There is no obvious LAR orthologue in the Arabidopsis genome, thus only an epicatechin was produced in Arabidopsis, rather than a dual catechin/epicatechin-based PAs similar to many other plants. In order to further investigate its function,35S:PtrLARl was introduced into Arabidopsis thaliana by floral dip method. HPLC analysis showed that epicatechin was not detected in wild-type Arabidopsis plants, while high concentration of epicatechin and catechin was accumulated in transgenic35S:PtrLAR1plants, confirming that PtrLAR1can simultaneously improve biosynthesis of catechin and epicatechin.In vitro assays showed that crude leaf extracts from35S:PtrLAR3transformants were able to inhibit significantly the hyphal growth of Marssonina brunnea f.sp. multigermtubi compared to the extracts from control plants. The transgenic35S:PtrLAR3poplar plants displayed a significant (P<0.05) reduction in their disease symptoms compared with the control. RT-PCR analysis showed that PtrLAR3expression was up-regulated in all transformants. These results suggested that constitutive expression of endogenous PtrLAR3could be exploited to improve resistance to fungal pathogens in poplar.