title: Improving total saccharification yield of Arabidopsis plants by vessel-specific complementation of caffeoyl shikimate esterase (cse) mutants.
abstract: BACKGROUND
  Caffeoyl 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.
  
  
  RESULTS
  Transactivation 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.
  
  
  CONCLUSIONS
  Our 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.
authors.affiliation: Department of Plant Systems Biology, VIB, 9052 Ghent, Belgium.|Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Ghent, Belgium.
authors.forename: Lívia
authors.initials: L
authors.lastname: Vargas
authors.affiliation: Department of Plant Systems Biology, VIB, 9052 Ghent, Belgium.|Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Ghent, Belgium.|Department of Botany, Institute of Biosciences, University of São Paulo, Butantã, SP Brazil.
authors.forename: Igor
authors.initials: I
authors.lastname: Cesarino
authors.affiliation: Department of Plant Systems Biology, VIB, 9052 Ghent, Belgium.|Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Ghent, Belgium.
authors.forename: Ruben
authors.initials: R
authors.lastname: Vanholme
authors.affiliation: Department of Plant Systems Biology, VIB, 9052 Ghent, Belgium.|Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Ghent, Belgium.
authors.forename: Wannes
authors.initials: W
authors.lastname: Voorend
authors.affiliation: Department of Plant Systems Biology, VIB, 9052 Ghent, Belgium.|Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Ghent, Belgium.
authors.forename: Marina
authors.initials: M
authors.lastname: de Lyra Soriano Saleme
authors.affiliation: Department of Plant Systems Biology, VIB, 9052 Ghent, Belgium.|Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Ghent, Belgium.
authors.forename: Kris
authors.initials: K
authors.lastname: Morreel
authors.affiliation: Department of Plant Systems Biology, VIB, 9052 Ghent, Belgium.|Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Ghent, Belgium.
authors.forename: Wout
authors.initials: W
authors.lastname: Boerjan
keywords: Arabidopsis thaliana; Caffeoyl shikimate esterase (CSE); Genetic engineering; Lignin; Saccharification; Secondary cell wall; Vessel-specific complementation