Simultaneous Down-Regulation of Dominant Cinnamoyl CoA Reductase and Cinnamyl Alcohol Dehydrogenase Dramatically Altered Lignin Content in Mulberry

Abstract

Mulberry (Morus alba L.) is a significant economic tree species in China. The lignin component serves as a critical limiting factor that impacts both the forage quality and the conversion efficiency of mulberry biomass into biofuel. Cinnamoyl CoA reductase (CCR; EC 1.21.1.44) and cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.95) are the key enzymes that catalyze the final two reductive steps in the biosynthesis of monolignols. In this study, we conducted a comprehensive functional analysis to validate the predominant CCR genes involved in monolignol biosynthesis. In this study, we initially validated the predominant CCR genes implicated in monolignol biosynthesis through an extensive functional analysis. Phylogenetic analysis, tissue-specific expression profiling and enzymatic assays indicated that MaCCR1 is the authentic CCR involved in lignin biosynthesis. Furthermore, the expression level of MaCCR1 exhibited a significant positive correlation with lignin content, and the down-regulation of MaCCR1 via virus-induced gene silencing resulted in altered lignin content in mulberry. The down-regulation of MaCCR1 and MaCAD3/4, both individually and concurrently, exhibited markedly different effects on lignin content and mulberry growth. Specifically, the simultaneous down-regulation of MaCCR1 and MaCAD3/4 significantly altered lignin content in mulberry, resulting in dwarfism of the plants. Conversely, the down-regulation of MaCAD3/4 alone not only decreased lignin content but also led to an increase in biomass. These findings offer compelling evidence elucidating the roles of MaCCRs in mulberry and identify specific target genes, thereby providing a crucial foundation for the genetic modification of lignin biosynthesis.

Keywords: Morus; VIGS; cinnamoyl CoA reductase; cinnamyl alcohol dehydrogenase; enzymatic assay; lignin.

Figure 1

Phylogenetic analysis of CCRs in different plants. Group I comprises genuine CCRs. MaCCRs are marked with full black circles. Sequence information is available in Table S3.

Figure 2

Alignment of MaCCRs and PtoCCR1 and 7. Populus tomentosa (Pto)CCR1 and PtoCCR7, which have been reported as genuine CCRs involved in lignin biosynthesis, were selected as reference CCRs. The red boxes indicate NAD(P)-binding and NADP specificity motifs, and the green boxes indicate CCR signature and substrate binding motifs (SBM). The red dots represent the catalytic triad.

Figure 3

Expression profiles of several MaCCRs. (A) Quantitative real-time PCR analysis of MaCCR1, (B) MaCCR3/4 and (C) MaCCR6 in different tissues. Data are presented as means ± SD of three biological replicates. One-way ANOVA and Tukey’s multiple comparisons test were performed. Different letters indicate significant differences (p < 0.05).

Figure 4

Optimum pH and temperature for MaCCR1 with feruloyl CoA as substrate. (A) The activity of MaCCR1 at different pH; (B) The activity of MaCCR1 at different temperatures. Data are presented as means ± SD of three replicates.

Figure 5

Knock-down of MaCCRs in mulberry stems and leaves and the corresponding lignin content. (A–F) Expression of MaCCR1, MaCCR3/4 and MaCCR6 in mulberry stem and leaf after VIGS treatment; (G–L) Lignin content in stems and leaves of mulberry trees after MaCCR1, MaCCR3/4 and MaCCR6 were knocked down. The lignin contents were measured and normalized to CK. Mulberry plants treated with TRV2 and TRV1 empty vectors were used as controls. VIGS lines for MaCCR1: VIGS-CCR1-1, VIGS-CCR1-5 and VIGS-CCR1-7; VIGS lines for MaCCR3/4: VIGS-CCR3/4-3, VIGS-CCR3/4-4 and VIGS-CCR3/4-6; VIGS lines for MaCCR6: VIGS-CCR6-1, VIGS-CCR6-7 and VIGS-CCR9-7. Data are presented as means ± SD. One-way ANOVA and Tukey’s multiple comparisons test were performed. Significant analysis with * (0.01 < p < 0.05), ** (0.005 < p < 0.01), *** (0.0001 < p < 0.005), **** (p < 0.0001).

Figure 6

Correlation of MaCCR expression levels with lignin contents in mulberry. (A−C) The red line indicates the correlation between lignin content and MaCCR1, MaCCR3/4 and MaCCR6 gene expression level in leaves. Biological repeats are represented by red squares. The black line indicates the correlation between lignin content and MaCCR1, MaCCR3/4 and MaCCR6 gene expression level in stems. Biological repeats are represented by black squares. Significant analysis with * (0.01 < p < 0.05), ** (0.005 < p < 0.01), *** (0.0001 < p < 0.005), **** (p < 0.0001).

Figure 7

Knock-down of MaCAD3/4 and MaCCR1 in mulberry stems and leaves. (A–D) Expression of MaCAD3/4 and MaCCR1 in mulberry stem and leaf after treatment. (E) Mulberry phenotypes of VIGS-MaCCR1, VIGS-MaCAD3/4, VIGS-MaCCR1+MaCAD3/4 and CK lines. Plants were incubated for 16 h/8 h in an artificial climate incubator. The scale bar indicating 5 cm is shown as a red line; data are presented as means ± SD of three biological replicates. One-way ANOVA and Tukey’s multiple comparisons test were performed. * (0.01 < p < 0.05), ** (0.005 < p < 0.01), *** (0.0001 < p < 0.005), **** (p < 0.0001).