Silencing CAFFEOYL SHIKIMATE ESTERASE Affects Lignification and Improves Saccharification in Poplar.

Caffeoyl shikimate esterase (CSE) was recently shown to play an essential role in lignin biosynthesis in Arabidopsis (Arabidopsis thaliana) and later in Medicago truncatula However, the general function of this enzyme was recently questioned by the apparent lack of CSE activity in lignifying tissues of different plant species. Here, we show that down-regulation of CSE in hybrid poplar (Populus tremula × Populus alba) resulted in up to 25% reduced lignin deposition, increased levels of p-hydroxyphenyl units in the lignin polymer, and a relatively higher cellulose content. The transgenic trees were morphologically indistinguishable from the wild type. Ultra-high-performance liquid chromatography-mass spectrometry-based phenolic profiling revealed a reduced abundance of several oligolignols containing guaiacyl and syringyl units and their corresponding hydroxycinnamaldehyde units, in agreement with the reduced flux toward coniferyl and sinapyl alcohol. These trees accumulated the CSE substrate caffeoyl shikimate along with other compounds belonging to the metabolic classes of benzenoids and hydroxycinnamates. Furthermore, the reduced lignin amount combined with the relative increase in cellulose content in the CSE down-regulated lines resulted in up to 62% more glucose released per plant upon limited saccharification when no pretreatment was applied and by up to 86% and 91% when acid and alkaline pretreatments were used. Our results show that CSE is not only important for the lignification process in poplar but is also a promising target for the development of improved lignocellulosic biomass crops for sugar platform biorefineries.

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

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.

Drought tolerance conferred to sugarcane by association with Gluconacetobacter diazotrophicus: a transcriptomic view of hormone pathways.

Sugarcane interacts with particular types of beneficial nitrogen-fixing bacteria that provide fixed-nitrogen and plant growth hormones to host plants, promoting an increase in plant biomass. Other benefits, as enhanced tolerance to abiotic stresses have been reported to some diazotrophs. Here we aim to study the effects of the association between the diazotroph Gluconacetobacter diazotrophicus PAL5 and sugarcane cv. SP70-1143 during water depletion by characterizing differential transcriptome profiles of sugarcane. RNA-seq libraries were generated from roots and shoots of sugarcane plants free of endophytes that were inoculated with G. diazotrophicus and subjected to water depletion for 3 days. A sugarcane reference transcriptome was constructed and used for the identification of differentially expressed transcripts. The differential profile of non-inoculated SP70-1143 suggests that it responds to water deficit stress by the activation of drought-responsive markers and hormone pathways, as ABA and Ethylene. qRT-PCR revealed that root samples had higher levels of G. diazotrophicus 3 days after water deficit, compared to roots of inoculated plants watered normally. With prolonged drought only inoculated plants survived, indicating that SP70-1143 plants colonized with G. diazotrophicus become more tolerant to drought stress than non-inoculated plants. Strengthening this hypothesis, several gene expression responses to drought were inactivated or regulated in an opposite manner, especially in roots, when plants were colonized by the bacteria. The data suggests that colonized roots would not be suffering from stress in the same way as non-inoculated plants. On the other hand, shoots specifically activate ABA-dependent signaling genes, which could act as key elements in the drought resistance conferred by G. diazotrophicus to SP70-1143. This work reports for the first time the involvement of G. diazotrophicus in the promotion of drought-tolerance to sugarcane cv. SP70-1143, and it describes the initial molecular events that may trigger the increased drought tolerance in the host plant.

Genomic evolution and complexity of the Anaphase-promoting Complex (APC) in land plants.

BACKGROUND: The orderly progression through mitosis is regulated by the Anaphase-Promoting Complex (APC), a large multiprotein E3 ubiquitin ligase that targets key cell-cycle regulators for destruction by the 26 S proteasome. The APC is composed of at least 11 subunits and associates with additional regulatory activators during mitosis and interphase cycles. Despite extensive research on APC and activator functions in the cell cycle, only a few components have been functionally characterized in plants. RESULTS: Here, we describe an in-depth search for APC subunits and activator genes in the Arabidopsis, rice and poplar genomes. Also, searches in other genomes that are not completely sequenced were performed. Phylogenetic analyses indicate that some APC subunits and activator genes have experienced gene duplication events in plants, in contrast to animals. Expression patterns of paralog subunits and activators in rice could indicate that this duplication, rather than complete redundancy, could reflect initial specialization steps. The absence of subunit APC7 from the genome of some green algae species and as well as from early metazoan lineages, could mean that APC7 is not required for APC function in unicellular organisms and it may be a result of duplication of another tetratricopeptide (TPR) subunit. Analyses of TPR evolution suggest that duplications of subunits started from the central domains. CONCLUSIONS: The increased complexity of the APC gene structure, tied to the diversification of expression paths, suggests that land plants developed sophisticated mechanisms of APC regulation to cope with the sedentary life style and its associated environmental exposures.