In this study, we used a candidate gene approach to identify the CAD and COMT genes involved in monolignol biosynthesis in B. distachyon. While well studied as key enzymes in the lignin pathway that influence forage quality, understanding of these enzymes is now of increased interest due to an apparently similar effect on biofuel feedstock quality
. To date, there have only been a few reports of CAD or COMT downregulation in transgenic monocots
[36–39]. These species, such as F. arundinacae and L. perenne have proven to be challenging research subjects. We report here the effectiveness of artificial microRNA silencing in a species considerably more amenable to research, B. distachyon, from which gained knowledge can be contributed towards the optimization of bioenergy grasses.
In monocots such as Z. mays, S. bicolor and O. sativa, CAD tends to exist as a multi-gene family with one CAD primarily involved in monolignol biosynthesis
[48, 49, 55]. In contrast, in the eudicot A. thaliana, the last step of monolignol biosynthesis is controlled synergistically by two genes, AtCAD-C and AtCAD-D. Based on amino acid sequence and gene expression pattern, we identified BdCAD1 as a likely candidate for having a role in lignin biosynthesis. Albeit with significantly lower efficiency than BdCAD1, Bradi4g29770 can also use coniferyl aldehyde and coniferyl alcohol as substrates and may also have a role in monolignol biosynthesis
. Unlike the other BdCADs, BdCAD1 contains all sequence features characteristic of a zinc-dependent alcohol dehydrogenase and more specifically, appears to be a member of the medium-chain dehydrogenase/reductase superfamily. High similarity in sequence motifs and substrate binding position suggests that BdCAD1 shares the same function as the bona fide CADs
. Consistent with multi-gene CAD families in Z. mays, S. bicolor and O. sativa, one particular protein in B. distachyon shared a significantly higher degree of homology with other known CADs than any other family member
[48, 49, 55]. Gene expression analysis revealed that BdCAD1 was the most highly expressed member of the gene family and that transcript abundance was particularly high in stem and root, where secondary cell walls are prevalent. Lignin-associated CAD expression was similarly high in stem and root tissues in P. virgatum, F. arundinacae, O. sativa, and S. bicolor[22, 38, 55].
The downregulation of BdCAD1 caused phenotypes characteristic of lignin deficiency without reducing plant biomass. The delay in flowering time of the amiR-cad1 plants is consistent with the phenotypes in five S. bicolor bmr mutants including CAD impaired bmr6[42, 59]. The variation in flowering time observed in lignin mutants from S. bicolor and B. distachyon reinforces the possibility of an evolutionarily conserved mechanism between cell wall biosynthesis and production of flowers
. Because lignin has a significant role in xylem function, it is possible that changes in lignin may alter development by perturbing water transport. However, this rationale seems unlikely considering that, while amiR-cad1 plants were developmentally delayed, mature transgenic plants were significantly larger than empty vector control plants (Figure
6D). An increase in aboveground stem biomass, even coupled with delayed flowering, is a favorable trait for a perennial energy crop, considering crop rotation will not be part of cultivation. While mutations in CAD can sometimes lead to pleiotropic effects of dwarfing, lodging, and a decrease in grain and/or biomass yield, these effects are mostly background-dependent
. Further understanding of gene-by-gene interactions that result in these deleterious effects will increase the efficiency of cultivar development.
We present here the brown midrib leaf phenotype for the first time in a C3 grass
. Genetic redundancy may explain why the phenotype has not been observed in mutant polyploid species such as T. aestivum and F. arundinacae; however, CAD mutants of diploid O. sativa do not exhibit a leaf brown midrib
[25, 55, 61, 62]. It has been suggested that the brown midrib phenotype may present itself differently in various species. In O. sativa, a mutation in GOLD HULL AND INTERNODE 2, which encodes a CAD enzyme, caused a red-brown pigment in the hull, internode, and basal leaf sheath while the leaf midrib did not show the same discoloration
. Similarly, recent research in B. distachyon indicated that CAD mutants displayed the red-brown pigmentation in various tissues including nodes and flowers, but not in the leaf midrib
. We did not observe color differences in tissues other than leaf midrib. In other species, including Populus sp. and N. tabacum, transverse stem cross-sections of transgenic CAD-downregulated plants exhibited unusually red xylem
[13, 16, 20]. On the other hand, no visible mutant phenotype was observed in CAD-RNAi Z. mays plants
. Here, the BdCAD1-downregulated plants phenotypically resembled Z. mays, S. bicolor, and P. glaucum leaf brown-midrib mutants. The brown-midrib phenotype may occur only when CAD activity is decreased beneath a certain threshold
. For example, in four lines of antisense-CAD transgenic tobacco with residual CAD activity ranging from 8-56%, the extent of CAD downregulation was correlated with the presence and pattern of reddish-brown xylem
. We measured relatively small changes in CAD activity, but at a developmental stage that had not yet exhibited the brown-midrib phenotype. Nonetheless, the amiR-cad1-8 plants which were most reduced in CAD activity were significantly more digestible when the plant had completely senesced. It is possible at a subsequent developmental stage characterized by greater lignin biosynthesis that diminished CAD activity would be more evident.
The quality of lignin was altered in amiR-cad1 plants, as indicated by a significant decrease in S units observed by thioacidolysis and in agreement with histochemical staining by the Maule reagent. Previous research in CAD-downregulated N. tabacum and CAD mutant B. distachyon showed that the most dramatic change in lignin composition in plants was a severe decrease in S lignin
[16, 64]. Some CAD gene knockouts produce functional lignin through increased incorporation of cinnamyl aldehyde subunits into the lignin polymer
. Previous reports for Z. mays, N. tabacum, and Populus sp. demonstrated an incorporation of aldehydes into the lignin polymer, in which increased coniferyl aldehyde caused an increase in intensity of the Wiesner stain
[13, 20]. On the contrary, the Wiesner stain in amiR-cad1 lines was less intense than control plants. A likely explanation is that the inhibition of S lignin synthesis still caused an accumulation of aldehydes, but specifically sinapyl aldehydes, which are not detected by Wiesner staining. This is consistent with decreased Wiesner staining and incorporation of 8-O-4-coupled sinapyl aldehyde in B. distachyon CAD mutant plants
Consistent with reports in N. tabacum, M. sativa, E. camaldulensis, Populus sp., F. arundinacae, and Z. mays, amiR-cad1 plants were unchanged in the amount of acetyl bromide soluble lignin polymer, but thioacidolysis indicated changes in lignin monomer composition
[13, 16–18, 20, 21, 24, 38]. The lignin in CAD downregulated plants was generally more reactive. This has been illustrated by improved pulping properties in Populus sp., forage digestibility in N. tabacum and F. arundinacae, saccharification in P. virgatum and B. distachyon, and digestibility in Z. mays[13, 16–18, 20, 22, 23, 25, 64]. Along the same lines, the modified lignin in amiR-cad1-8 improved biological conversion efficiency by a statistically significant 17%.
One protein among the four COMTs identified in B. distachyon, BdCOMT4 (Bradi3g16530), contained all of the signature features of a plant O-methyltransferase. Plant O-methyltransferases tend to have broad substrate specificity, and all nine substrate binding and positioning residues in BdCOMT4 are common to COMT proteins in other species. Similar to L. perenne, F. arundinacae, P. tremuloides, and M. sativa COMT genes
[38, 66–68], BdCOMT4 was the most highly expressed COMT in stem, root, and leaf tissues than any of the three other B. distachyon family members. Similar to observations made in P. tremuloides, BdCOMT4 expression was relatively low in leaves compared to stems
. Phylogeny, amino acid sequence, and the abundance of transcript in lignified tissues concurrently support that BdCOMT4 is an O-methyltransferase involved in monolignol biosynthesis in B. distachyon.
The downregulation of COMT resulted in changes in various phenotypic traits. Mutants tended to flower earlier than the empty vector control, as seen for the lignin mutants bm1 in Z. mays and bmr7 in S. bicolor[42, 59]. In general, the brown midrib phenotype is not common in COMT-downregulated transgenic plants, with the only reports of a reddish-brown coloration of the leaf and internodes being in Z. mays[37, 69]. Although the cause of discoloration in plants with impaired CAD activity is often attributed to the incorporation of aldehydes into the lignin polymer, there is no obvious correlation between the phenotype and the activities of other enzymes of the monolignol biosynthesis pathway. Previous biochemical analysis has indicated that the brown coloration is not a result of accumulated carotenoids, anthocyanins, flavones, tannins, or flavonols, but could possibly be due to incorporation of other phenolic compounds into the lignin polymer
. Accumulation of novel 5-OH-G units has been observed in COMT-downregulated transgenic M. sativa and Z. mays, although a visual phenotype associated with this phenomenon has not been defined
[33, 37, 69, 70].
In our transgenic B. distachyon, the perturbation of the COMT enzyme had a deleterious effect on the total quantity of lignin produced in the plant. Similarly, downregulated COMT mutants in Z. mays, F. arundinacae, L. perenne, S. bicolor, and Saccharum spp. were also reduced in total lignin
[38, 39, 69, 71]. Staining with the Maule reagent revealed an obvious difference between control and amiR-comt4 transverse stem cross sections. We measured an increase in ethanol yield of up to 10% in amiR-comt4 lines, which is consistent with the characterization of COMT mutants in other species
[33, 37, 38, 69, 71–73].