Phenolic compounds and expression of 4CL genes in silver birch clones and Pt4CL1a lines

Abstract

A small multigene family encodes 4-coumarate:CoA ligases (4CLs) catalyzing the CoA ligation of hydroxycinnamic acids, a branch point step directing metabolites to a flavonoid or monolignol pathway. In the present study, we examined the effect of antisense Populus tremuloides 4CL (Pt4CL1) to the lignin and soluble phenolic compound composition of silver birch (Betula pendula) Pt4CL1a lines in comparison with non-transgenic silver birch clones. The endogenous expression of silver birch 4CL genes was recorded in the stems and leaves and also in leaves that were mechanically injured. In one of the transgenic Pt4CL1a lines, the ratio of syringyl (S) and guaiacyl (G) lignin units was increased. Moreover, the transcript levels of putative silver birch 4CL gene (Bp4CL1) were reduced and contents of cinnamic acid derivatives altered. In the other two Pt4CL1a lines changes were detected in the level of individual phenolic compounds. However, considerable variation was found in the transcript levels of silver birch 4CLs as well as in the concentration of phenolic compounds among the transgenic lines and non-transgenic clones. Wounding induced the expression of Bp4CL1 and Bp4CL2 in leaves in all clones and transgenic lines, whereas the transcript levels of Bp4CL3 and Bp4CL4 remained unchanged. Moreover, minor changes were detected in the concentrations of phenolic compounds caused by wounding. As an overall trend the wounding decreased the flavonoid content in silver birches and increased the content of soluble condensed tannins. The results indicate that by reducing the Bp4CL1 transcript levels lignin composition could be modified. However, the alterations found among the Pt4CL1a lines and the non-transgenic clones were within the natural variation of silver birches, as shown in the present study by the clonal differences in the transcripts levels of 4CL genes, soluble phenolic compounds and condensed tannins.

Figure 1. Phylogenetic analysis of silver birch (Betula pendula) 4CL and 4CL-like sequences.

ML tree was constructed using the cds of silver birch putative 4CL (Bp4CL1, Bp4CL2, Bp4CL4) genes and 4CL-like (Bp4CL3) gene.

Figure 2. Relative expression of Bp4CL1-4 genes in mechanically wounded silver birch (Betula pendula) leaves.

The Bp4CL1 (A, B) and Bp4CL2 (C, D) expression in leaves collected immediately (w0) and 1, 3, 12, 24, 48, and 162 h after mechanical wounding of clones A and R and transgenic lines A1 and A5 (A, C) and clones E5382 and E5396 and transgenic lines E5382 and E5396 (B, D). Values represent mean ± SE calculated using the mean of control leaves as normalizer within each line/clone and PP2A as reference.

Figure 3. The contents of phenolic compounds and condensed tannins in silver birch (Betula pendula) stems and leaves.

Stems (A) and leaves (B) of clones A, E5382, E5396, and R, and Pt4CL1a lines A1, A5 and E5282/3 and nptII line E5396/4. Values are mg/DW g mean ± SE. Different letters above the columns denote significant (P<0.05) difference between the clones. Stars above the columns of transgenic lines denote significant (P<0.05) difference between transgenic line to non-transgenic clone.

Figure 4. Wounding effects on the leaf phenolics of silver birch (Betula pendula).

The contents of kaempferols (A, B), quercetin (C, D), and p-OH-cinnamic acid derivatives (E, F), total content of HPLC identified phenolics (G, H), soluble CT (I, J), total CT (K, L), (+)-catechin (M, N) and protocatechuic acid (O, P) concentrations in untreated (c) and mechanically wounded (w) leaves collected at day 0 and 21 d after treatment in clones A and R and Pt4CL1a lines A1 and A5 (A, C, E, G) and clones E5382 and E5396 and transgenic lines E5382/3 and E5396/4 (B, D, F, H). Values are mg/DW g mean ± SE.