Genetic engineering of Arabidopsis to overproduce disinapoyl esters, potential lignin modification molecules

Abstract

BackgroundMonolignol-like molecules can be integrated into lignin along with conventional monolignol units, and it has been shown that the incorporation of non-canonical subunits can be used to generate hydrolysable lignin by introduction of ester linkages into the polymer and that this type of lignin is more easily removable. Disinapoyl esters (DSEs), which to some degree resemble the monolignol sinapyl alcohol, may be promising lignin modifying units for this purpose. As a first step toward determining whether this goal is achievable, we manipulated metabolic flux in Arabidopsis to increase the amounts of DSEs by overexpressing sinapoylglucose:sinapoylglucose sinapoyltransferase (SST) which produces two main DSEs, 1,2-disinapoylglucose, and another compound we identify in this report as 3,4-disinapoyl-fructopyranose.ResultsWe succeeded in overproducing DSEs by introducing an SST-overexpression construct into the sinapoylglucose accumulator1 (sng1-6) mutant (SST-OE sng1-6) which lacks several of the enzymes that would otherwise compete for the SST substrate, sinapoyglucose. Introduction of cinnamyl alcohol dehydrogenase-c (cad-c) and cad-d mutations into the SST-OE sng1-6 line further increased DSEs. Surprisingly, a reduced epidermal fluorescence (ref) phenotype was observed when SST-OE sng1-6 plants were evaluated under UV light, which appears to have been induced by the sequestration of DSEs into subvacuolar compartments. Although we successfully upregulated the accumulation of the target DSEs, we did not find any evidence showing the integration of DSEs into the cell wall.ConclusionsOur results suggest that although phenylpropanoid metabolic engineering is possible, a deeper understanding of sequestration and transport mechanisms will be necessary for successful lignin engineering through this route.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-017-0725-0) contains supplementary material, which is available to authorized users.

Cellular and Genetic Regulation of Coniferaldehyde Incorporation in Lignin of Herbaceous and Woody Plants by Quantitative Wiesner Staining

“…Arabidopsis thaliana plants were grown from seeds on 1:3 (v:v) vermiculite/soil and hybrid poplar in sterile magenta ™ boxes on 0.5 × MS medium (Duchefa, M0222.0050) with 0.4% phytagel (Sigma, P8169) in controlled growth chambers under a 16/8 h and 22°C/18°C photoperiod with 60% humidity and 150 µmol m −2 s −1 illumination. Arabidopsis mutants in the Columbia-0 background included: 4cl1-1 (SALK_142526; Van Acker et al, 2013), 4cl2-4 (SALK_110197; Li et al, 2015), ccr1-3 (SALK_123-689;Mir Derikvand et al, 2008), cad4 (cad-c; SAIL_1265_A06; Lee et al, 2017), cad5 (cad-d;SAIL_776_B06;Lee et al, 2017), ccoaomt1 (SALK_151507; Kai et al, 2008), fah1 (EMS mutant; Meyer et al, 1998), omt1 (SALK_135290; Tohge et al, 2007), and double mutants 4cl1-1x4cl2-4, ccr1-3xfah1 and cad4xcad5. All Arabidopsis plants were genotyped to select homozygous mutants using PCR with specific primer pairs ( Table S1).…”

Simultaneous regulation of F5H in COMT‐RNAi transgenic switchgrass alters effects of COMT suppression on syringyl lignin biosynthesis

“…Lignin engineering is currently focusing on incorporation of atypical lignin monomers in plant cell walls for improving the digestibility of lignocellulosic biomass (Lee et al ., ). It has been indicated that down‐regulation of F5H in Arabidopsis cinnamyl alcohol dehydrogenase ( CAD ) double mutant ( cadc and cadd ) can produce lignins derived exclusively from polymerization of coniferaldehyde (Anderson et al ., ).…”

Arabidopsis Thaliana: From Weed to Model Organism

“…Arabidopsis thaliana is a low‐cost model organism amenable to several areas of research of pressing global importance. These research areas include climate change (Ahuja, de Vos, Bones, & Hall, ; Hancock et al., ; Li, Huang, Bergelson, Nordborg, & Borevitz, ; Samis et al., ; Wilczek, Cooper, Korves, & Schmitt, ), food security (Blümel, Dally, & Jung, ; Chardon, Noël, & Masclaux‐Daubresse, ; Chew & Halliday, ; Lenk, Wenig, Mengel, Häußler, & Vlot, ; Naqvi et al., ; O'Conner et al., ), biofuel production (Lee, Mo, Kim, & Chapple, ; Vargas et al., ), and vaccine development (Kalbina, Engstrand, Andersson, & Strid, ; Sohn et al., ).…”