Improving total saccharification yield of Arabidopsis plants by vessel-specific complementation of caffeoyl shikimate esterase (cse) mutants

Abstract

BackgroundCaffeoyl shikimate esterase (CSE) was recently characterized as an enzyme central to the lignin biosynthetic pathway in Arabidopsis thaliana. The cse-2 loss-of-function mutant shows a typical phenotype of lignin-deficient mutants, including collapsed vessels, reduced lignin content, and lignin compositional shift, in addition to a fourfold increase in cellulose-to-glucose conversion when compared to the wild type. However, this mutant exhibits a substantial developmental arrest, which might outweigh the gains in fermentable sugar yield. To restore its normal growth and further improve its saccharification yield, we investigated a possible cause for the yield penalty of the cse-2 mutant. Furthermore, we evaluated whether CSE expression is under the same multi-leveled transcriptional regulatory network as other lignin biosynthetic genes and analyzed the transcriptional responses of the phenylpropanoid pathway upon disruption of CSE.ResultsTransactivation analysis demonstrated that only second-level MYB master switches (MYB46 and MYB83) and lignin-specific activators (MYB63 and MYB85), but not top-level NAC master switches or other downstream transcription factors, effectively activate the CSE promoter in our protoplast-based system. The cse-2 mutant exhibited transcriptional repression of genes upstream of CSE, while downstream genes were mainly unaffected, indicating transcriptional feedback of CSE loss-of-function on monolignol biosynthetic genes. In addition, we found that the expression of CSE under the control of the vessel-specific VND7 promoter in the cse-2 background restored the vasculature integrity resulting in improved growth parameters, while the overall lignin content remained relatively low. Thus, by restoring the vascular integrity and biomass parameters of cse-2, we further improved glucose release per plant without pretreatment, with an increase of up to 36 % compared to the cse-2 mutant and up to 154 % compared to the wild type.ConclusionsOur results contribute to a better understanding of how the expression of CSE is regulated by secondary wall-associated transcription factors and how the expression of lignin genes is affected upon CSE loss-of-function in Arabidopsis. Moreover, we found evidence that vasculature collapse is underlying the yield penalty found in the cse-2 mutant. Through a vessel-specific complementation approach, vasculature morphology and final stem weight were restored, leading to an even higher total glucose release per plant.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0551-9) contains supplementary material, which is available to authorized users.

Silencing CAFFEOYL SHIKIMATE ESTERASE Affects Lignification and Improves Saccharification in Poplar

“…Although the underlying molecular mechanisms that result in developmental arrest in lignin-deficient plants are largely unknown, it is likely that they depend on the particular step of the pathway that is blocked (Bonawitz and Chapple, 2013). Recently, we found evidence that vasculature collapse underlies the yield penalty found in the Arabidopsis cse-2 mutant, as restoring CSE expression specifically in vessels also restored vasculature morphology and final stem weight, leading to an even higher total Glc release per plant (Vargas et al, 2016). In poplar, CSE down-regulation resulted in higher amounts of Glc released upon saccharification with and without pretreatment, which is likely associated with reduced lignin amounts combined with increased relative cellulose contents.…”

Vessel-Specific Reintroduction of CINNAMOYL-COA REDUCTASE1 (CCR1) in Dwarfed ccr1 Mutants Restores Vessel and Xylary Fiber Integrity and Increases Biomass

“…Previous vessel-complementation attempts using VND6 and VND7 promoter sequences did not achieve a full restoration of vessel integrity and growth while maintaining the high sugar yield for Arabidopsis plants mutated in genes involved in lignin and hemicellulose biosynthesis (Petersen et al, 2012;Yang et al, 2013;Vargas et al, 2016). This could be the consequence of (1) the targeted cell wall biosynthesis gene or (2) the expression level and pattern conferred by the chosen promoter.…”

“…To fully restore the integrity of the vessels and the growth of ccr1 mutants, a vessel-specific promoter was required that conferred higher expression levels and/or a broader expression pattern than the previously tested VND6 and VND7 promoters (Petersen et al, 2012;Yang et al, 2013;Vargas et al, 2016). The artificial ProSNBE has been shown to direct the expression of reporter genes to xylem vessel cells of the Arabidopsis inflorescence stem (McCarthy et al, 2011).…”

“…Moreover, these lines had lower sugar yields per mg of cell wall when compared with c4h mutants. A similar strategy has been used in Arabidopsis cse-2 mutants (Vargas et al, 2016). Here, introducing CSE under the control of a 1,004-bp VND6 or 1,997-bp VND7 promoter sequence partially restored their growth and vascular integrity.…”

“…In these studies, VASCULAR-RELATED NAC DOMAIN6 (VND6) and VND7 promoter sequences were used to drive the expression of a lignin biosynthesis gene in the respective lignin mutant, thereby aiming at reintroducing lignin biosynthesis specifically in vessel cells. The expression of VND6 has been shown to be restricted to the metaxylem vessels, whereas VND7 had the highest expression level in protoxylem vessels (Kubo et al, 2005;Zhong et al, 2008;Vargas et al, 2016). An example of this strategy includes the partial restoration of the dwarfed phenotype of c4h knockdown mutants by the reintroduction of the C4H gene under the control of a 2,757-bp VND6 promoter sequence, resulting in plants with normal, open vessels (Yang et al, 2013).…”