Characterization and functional analysis of the Hydroxycinnamoyl-CoA: shikimate hydroxycinnamoyl transferase (HCT) gene family in poplar

Abstract

Hydroxycinnamoyl-CoA: shikimate hydroxycinnamoyl transferase (HCT) divides the mass flux to H, G and S units in monolignol biosynthesis and affects lignin content. Ten HCT homologs were identified in the Populus trichocarpa (Torr. & Gray) genome. Both genome duplication and tandem duplication resulted in the expansion of HCT orthologs in Populus. Comprehensive analysis including motif analysis, phylogenetic analysis, expression profiles and co-expression analysis revealed the divergence and putative function of these candidate PoptrHCTs. PoptrHCT1 and 2 were identified as likely involved in lignin biosynthesis. PoptrHCT9 and 10- are likely to be involved in plant development and the response to cold stress. Similar functional divergence was also identified in Populus tomentosa Carr. Enzymatic assay of PtoHCT1 showed that PtoHCT1 was able to synthesize caffeoyl shikimate using caffeoyl-CoA and shikimic acid as substrates.

Keywords: Divergence; Enzymatic synthesis; Gene family; Hydroxycinnamoyl- CoA: shikimate hydroxycinnamoyl transferase; Monolignol; Populus.

Figure 1. Schematic diagram of reaction catalyzed by HCT in monolignol biosynthesis pathway.

R=Shikimate. 4CL, 4-coumarate-CoA ligase; C3H, p- coumarate 3-hydroxylase; HCT, Hydroxycinnamoyl-CoA: shikimate hydroxycinnamoyl transferase. Compounds in red shadow are precursors for H units and in green shadow are for G and S units.

Figure 2. PoptrHCT orthologs organization and phylogenetic analysis.

(A) Organization of HCT orthologs on Populus chromosomes. Regions that are assumed to correspond to homologous genome blocks are shaded gray and connected by lines. The position of genes is indicated with an arrowhead. (B) Phylogenetic analysis of HCT homologs from Populus trichocarpa and other plant species. The PoptrHCT1 and 2 were marked with full black triangle. Two groups for HCT orthologs were shown and HCTs in Group I are likely to transfer hydroxycinnamates to shikimate and have been implicated in monolignol biosynthesis. The scale bar indicates 0.5 amino acid substitutions per site in given length. The accession numbers of sequences used are as followed: Arabidopsis thialiana AtHCT (AT5G48930), AtHCTlike (AT4G29250); Amborella trichopoda AtrHCT1 (ATR_00137G00320), AtrHCT2 (ATR_00727G00010); Cynara cardunculus CcaHCT (DQ104740), CcaHQT(ABK79690); Lycopersicon esculentum LeHQT (AJ582652); Larix kaempferi LkaHCT (AHA44839); Nicotiana tabacum NtaHCT (Q8GSM7), NtaHQT (CAE46932); Picea lauca PglHCT (CZO01061061); Populus trichocarpa PoptrHCT1 (PT01G04290), PoptrHCT2 (PT03G18390), PoptrHCT3 (PT05G02800), PoptrHCT4 (PT05G02810), PoptrHCT5 (PT05G02840), PoptrHCT6 (PT18G03270), PoptrHCT7 (PT18G10470), PoptrHCT8 (PT18G10480), PoptrHCT9 (PT18G10540), PoptrHCT10 (PT18G10550); Physcomitrella patens PpHCT1 (PP00022G00830); Panicum virgatum PviHCT1a (JX845714), PviHCT2 (KC696573 ); Sorghum bicolor SbHCT (XP_002452435.1).

Figure 3. Alignment of PoptrHCT and PoptrHCT orthologs compared to shikimate-specific HCTs from Arabidopsis and sorghum and HQT from tomato.

Red full stars indicate shikimate binding sites, red full circles indicate carbonyl group of p-coumaroyl moiety binding sites, purple full triangle indicate carbonyl group of shikimate moiety binding sites and the blue full circles indicate sites involved in catalysis. Accessions are as in Fig. 2. Detailed references are also available in Table 1.

Figure 4. Expression profile and co-expression network of HCT orthologs in poplar.

(A). Expression profile of HCT orthologs in Populus. Tissues or specific parts of plants are indicated with the respective abbreviations: WS, whole stems; BM, Bark and mature phloem; C, cambium; DP, developing phloem; DX, developing xylem; ML, mature leaf; SLp, shoot and leaf primordium. (B) Expression profile of HCT orthologs in P. tomentosa. (C) Co-expression network of PoptrHCT orthologs with identified genes involved in lignin biosynthesis. The support information is available in Table S3. Only nodes with Pearson correlation coefficients ¿0.9 were shown and considered as close co-expression.

Figure 5. PtoHCT1 catalyzes enzymatic synthesis of caffeoyl shikimate.

LC separation of reactions with MS detection (selected ion signals) (A) at initiation of the reaction (B) after 80s or (C) after 120s.

Figure 6. The structure of PtoHCT1 and docking with caffeoyl-CoA.

(A) Structure alignment of AtHCT (green) and PtoHCT (purple). (B) PtoHCT docked with caffeoyl-CoA. Blue ligand is caffeoyl-CoA and active sites are labeled.