Pathway-specific genetic pretreatment strategy to improve bioenergy feedstock

The plant secondary wall is composed of a complex mixture of cellulose, hemicellulose (e.g., xylan), and lignin. The transcription factor MYB46 (At5g12870) has been reported as a central regulator of the secondary wall formation. However, constitutive overexpression of MYB46 results in a severe growth penalty, possibly due to ectopic lignification in the parenchymatous and photosynthetic cells. To test this hypothesis, we constitutively overexpressed MYB46 in the genetic background of a T-DNA insertion mutant 4cl1-2 of 4-coumarate-CoA ligase 1 (4CL1), a key lignin biosynthesis gene. As expected, the transgenic Arabidopsis plants 35S::MYB46/4cl1-2 grow normally, similarly to the 4cl1-2 mutant, with elevated expressions of three secondary wall cellulose synthase genes and xylan biosynthesis genes. Consistent with the gene expression changes, the immunohistological staining of stem tissues showed the increased accumulation of both cellulose and xylan contents in the 35S::MYB46/4cl1-2 plants. Further biochemical analyses confirmed that 35S::MYB46/4cl1-2 plants have higher level accumulations of crystalline cellulose and xylan in both leaf and stem tissues than wild-type control plants as well as the 4cl1-2 mutant, up to 1.18- and 1.13-fold respectively, with considerably reduced lignin content (up to 0.40-fold). Subsequent analysis of enzymatic glucose release from cell wall materials revealed that 35S::MYB46/4cl1-2 plants have higher yields up to 1.27-fold increase in both leaf and stem tissues than that of control plants and 4cl1-2 mutant. Our results showed that overexpression of MYB46 with simultaneous reduction of lignin biosynthesis significantly increases both fermentable sugar contents and cell wall digestibility without growth penalty associated with MYB46 overexpression.