Simultaneous Downregulation of MTHFR and COMT in Switchgrass Affects Plant Performance and Induces Lesion-mimic Cell Death

Switchgrass (Panicum virgatum) has been developed into a model lignocellulosic bioenergy crop. Downregulation of caffeic acid O-methyltransferase (COMT), a key enzyme in lignin biosynthesis, led to altered lignification and increased biofuel yield in switchgrass. Methylenetetrahydrofolate reductase (MTHFR) mediated C1 metabolism provides methyl units consumed by COMT. It was predicted that co-silencing of MTHFR and COMT would have a more significant impact on lignification than either of the single genes. However, our results showed that strong downregulation of MTHFR in a COMT-deficient background led to altered plant growth and development, but no significant change in lignin content or composition was found when compared with COMT plants. Another unexpected finding is that the double MTHFR/COMT downregulated plants showed a novel lesion-mimic leaf phenotype. Molecular analyses revealed that the lesion-mimic phenotype was caused by the synergistic effect of MTHFR and COMT genes, with MTHFR playing a predominant role. Microarray analysis showed significant induction of genes involved in oxidative and defenserelated processes. The results demonstrated the lack of additive effects of MTHFR and COMT on lignification. Furthermore, this research revealed an unexpected role of the two genes in the modulation of lesion-mimic cell death as well as their synergetic effects on agronomic performance. High-quality total RNA from duplicate biological replicates of the transgenic lines MT/CO-37 and MT/CO-47 (severe phenotype group), MT/CO-38 and MT/CO-67 (moderate phenotype group) was isolated from young leaves (Leaf 4) at E4 stage using Spectrum157™ Plant Total RNA Kit (Sigma-Aldrich). To eliminate background differences among transgenics, except wild-type plant, MT/CO-60 with no more than 40% decrease of both MTHFR and COMT genes was also selected as a control. Leaf 4 at E4 stage was used because the lesion phenotype at this stage was fully emerged in leaf 2, half developed in leaf 3, but couldn’t be observed in leaf 4. RNA amplification, labelling and hybridization were done as described by Fu et al. (2012).